Free
Pain Medicine  |   February 2010
Direct Evidence for the Ongoing Brain Activation by Enhanced Dynorphinergic System in the Spinal Cord under Inflammatory Noxious Stimuli
Author Affiliations & Notes
  • Yasuko Taketa, M.D., Ph.D.
    *
  • Keiichi Niikura, Ph.D.
  • Yasuhisa Kobayashi, M.Sc.
  • Masaharu Furuya, B.S.
  • Toshikazu Shimizu, M.Sc.
  • Michiko Narita, B.S.
    §
  • Satoshi Imai, Ph.D.
  • Naoko Kuzumaki, Ph.D.
  • Yoshie Maitani, Ph.D.
    #
  • Mitsuaki Yamazaki, M.D., Ph.D.
    **
  • Eiichi Inada, M.D., Ph.D.
    ††
  • Masako Iseki, M.D., Ph.D.
    ‡‡
  • Tsutomu Suzuki, Ph.D.
    §§
  • Minoru Narita, Ph.D.
    ∥∥
  • * Research Associate, Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, and Department of Anesthesiology and Pain Medicine, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan. † Postdoctoral Fellow, ‡ Graduate Student, § Research Assistant, ∥ Research Associate, Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences. # Professor, Institute of Medicinal Chemistry, Hoshi University School of Pharmacy and Pharmaceutical Sciences. ** Professor, Department of Anesthesiology, Toyama University School of Medicine, Toyama, Japan. †† Professor, ‡‡ Associate Professor, Department of Anesthesiology and Pain Medicine, Juntendo University School of Medicine. §§ Professor, Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences. ∥∥ Associate Professor, Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Department of Anesthesiology and Pain Medicine, Juntendo University School of Medicine, and Department of Anesthesiology, Toyama University School of Medicine.
Article Information
Pain Medicine / Central and Peripheral Nervous Systems / Pain Medicine
Pain Medicine   |   February 2010
Direct Evidence for the Ongoing Brain Activation by Enhanced Dynorphinergic System in the Spinal Cord under Inflammatory Noxious Stimuli
Anesthesiology 2 2010, Vol.112, 418-431. doi:10.1097/ALN.0b013e3181ca31d9
Anesthesiology 2 2010, Vol.112, 418-431. doi:10.1097/ALN.0b013e3181ca31d9
What We Already Know about This Topic
  • ❖ Spinal dynorphin release contributes to hypersensitivity to stimuli in rodents, but whether it activates supraspinal structures related to pain is not known
What This Article Tells Us That Is New
  • ❖ Injection of complete Freund's adjuvant into the paw of mice increased dynorphin in the spinal cord and increased activity in several cortical regions associated with pain processing
  • ❖ These effects were not present in genetically altered mice lacking dynorphin
ENDOGENOUS opioid peptides are efficient analgesics that bind to opioid receptors. These peptides are typically produced to counteract chronic pain. They are classified as enkephalins, endorphins, and dynorphins.1 Dynorphins are neuropeptides that inhibit neuronal activity through κ-opioid receptors.2 Dynorphin A (1–17) is one of the major proteolytic fragments of prodynorphin2 and has been shown to be distributed widely throughout the central nervous system.2–5 Many experimental models of pathologic pain, including inflammatory pain,6–10 neuropathic pain,10–12 bone cancer pain,13 and abnormal pain (hyperalgesia),14 show a significant induction of dynorphin A in the spinal cord. Relatively low doses of dynorphin A produce analgesia by virtue of acting as an inhibitory opioid peptide and preferentially activating κ-opioid receptors.10,15,16 In contrast, high doses of dynorphin A elicit pronociceptive behaviors, such as biting, licking, and scratching,17 or allodynia.3,18,19 In such cases, the actions are not sensitive to opioid antagonist but are blocked by intrathecal pretreatment with MK-801, a noncompetitive antagonist of N  -methyl-d-aspartic acid (NMDA) receptor channels.19 Taken together, increased dynorphin A in the spinal cord may play an important role in the nociceptive state in terms of its paradoxical effects on neurotransmission.
Functional magnetic resonance imaging can be used to investigate spatial and temporal brain activation. Functional magnetic resonance imaging has been used to evaluate pain perception in the central nervous system in healthy humans and in those with various kinds of pain.20,21 Noxious heat stimulation in humans or repetitive heat stimulation through peltier elements in animals activates several brain regions, so-called pain matrix.22–25 Recent studies have demonstrated that neuroimaging in humans and animals can detect changes in regional activity initiated by the administration of drugs that induce or modulate pain, such as morphine, ketamine, formalin, and capsaicin.26–29 Furthermore, these studies have introduced the new term pharmacological functional magnetic resonance imaging (phMRI) for this technique, which promises to become an important new tool for the researcher who is interested in mapping and understanding the pain mechanism. We previously demonstrated that neuropathic pain-like transmission evoked by the spinal activation of protein kinase C caused a significant increase in the activity of several brain regions using a mouse phMRI assay.30 
In this study, we investigated whether a deletion of the gene that encodes the prodynorphin could affect the brain regions activated after unilateral intraplantar injection of complete Freund's adjuvant (CFA) injection using phMRI method in mice and whether direct intrathecal administration of dynorphin A (1–17) at relatively high doses could facilitate brain activity in regions that are associated with pain perception via  an NMDA receptor-mediated pathway.
Materials and Methods
Animals
This study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, Hoshi University, as adopted by the Committee on Animal Research of Hoshi University, which is accredited by the Ministry of Education, Culture, Sports, Science and Technology of Japan. This study was approved by the Animal Research Committee of Hoshi University. The first series of experiments using 41 mice, either wild-type (C57BL/6 and 129S4/SvJ mixed genetic background, 21 males, 8–10 weeks old, 18–23 g; The Jackson Laboratory, Bar Harbor, ME) or prodynorphin gene-knockout (C57BL/6 and 129/SvJ mixed genetic backgrounds, 20 males, 8–10 weeks old, 18–23 g; The Jackson Laboratory) mice was also performed. Ninety-seven male mice (8–10 weeks old, 18–23 g) were used for another series of experiments in C57BL/6J mice (CLEA Japan, Inc., Tokyo, Japan). Animals were kept in a room with an ambient temperature of 23°± 1° C and a 12-h light-dark cycle (lights on 8:00 am to 8:00 pm). Food and water were available ad libitum  . All animals were individually housed, and all behavioral studies were performed during the light period. At the end of the experiments, animals were humanely killed by a rising concentration of ethyl ether.
Intrathecal Injection
Intrathecal injection was performed as described by Hylden and Wilcox31 using a 25-μl Hamilton syringe with a 30 ½-gauge needle. The needle was inserted into the intervertebral space between L5 and L6 level of the spinal cord. A reflexive flick of the tail was considered to be an accuracy of each injection. The injection volume was 4 μ l for intrathecal injection.
Inflammatory Pain Model
A persistent inflammatory pain model was produced by unilateral intraplantar injection of CFA (mycobacterium tuberculosis; Sigma-Aldrich Co., St. Louis, MO) in a volume of 50 μ l into the plantar surface of the right hind paw (ipsilateral side) of mice during anesthesia with isoflurane.32–34 Control mice were given saline in a volume of 50 μ l into the plantar surface of the right hind paw.
Pain-like Behaviors
To observe the pain-like behaviors induced by the intrathecal injection of dynorphin A (1–17) in normal mice, groups of mice were individually placed in an observation cage immediately after intrathecal injection of dynorphin A (1–17) (0.3, 3, and 30 pmol/mouse; Peptide Institute, Inc., Osaka, Japan) or saline. Intrathecal pretreatment with MK-801 (0.3 or 1 nmol/mouse) was performed 30 min before the intrathecal injection of dynorphin A (1–17) (30 pmol/mouse).
To assess the pain-like behaviors induced by inflammatory pain, groups of mice were individually placed in an observation cage immediately after the intraplantar injection of CFA or saline. Intrathecal pretreatment with antiserum against dynorphin A (1–17) (1:100; Peninsula Laboratories, Inc., San Carlos, CA) or control serum (normal rabbit serum; Vector Laboratories, Inc., Burlingame, CA) was performed 30 min before the intraplantar injection of CFA.
The number of licking or flinching behaviors was counted, and the duration of licking and flinching behaviors was measured for 20 min after treatment.
Functional Imaging
Functional imaging was performed, as previously described.30 To investigate the effect of the intraplantar injection of CFA, prodynorphin gene-knockout mice or wild-type mice were anesthetized using isoflurane immediately after the intraplantar injection of CFA or vehicle. To investigate the effect of a single intrathecal treatment with dynorphin A (1–17) in C57BL/6J mice, mice were anesthetized using isoflurane immediately after intrathecal injection of dynorphin A (1–17) (30 pmol/mouse). Animals were then transferred to a cradle that was designed to fit inside the probe of the magnetic resonance system and supplied with 1% isoflurane via  a fitted mask. A continuous phMRI scanning protocol was used to study the changes in brain signal intensity using T2 star-weighted blood oxygenation level-dependent (BOLD) contrast. BOLD responses were measured hourly from 30 min to 6 h at all brain levels.
Experiments were performed with a Unity Inova spectrometer (Varian, Palo Alto, CA) that was interfaced to a 9.4-T/31-cm horizontal bore magnet equipped with actively shielded gradients capable of 300 mT/m in a rise time of 500 s (Magnex Scientific, Abingdon, United Kingdom). High-resolution anatomical scans were collected using a fast spin echo pulse sequence (repetition time = 2000 ms, echo time = 45 ms, field of view = 25.6 × 25.6 mm2, 1. 0-mm slice thickness, 256 × 256 data matrix).
Functional images were obtained with the two-slice gradient-echo fast imaging sequence (echo time = 25 ms, repetition time = 70 ms, 30-° flip angle, 128 × 128).35 One-millimeter-thick slices were simultaneously acquired over a field of view of 25.6 mm2, number of average of 2, and an acquisition time of 32 s. Typically, 3–6 images were collected at baseline, followed by intrathecal injection of dynorphin A (1–17) or intraplanter injection of CFA. Intrathecal pretreatment with MK-801 (1 nmol/mouse) was performed 30 min before the intrathecal injection of dynorphin A (1–17). The regions of interest were selected, and statistical analyses were performed using the image-analysis software ImageJ (National Institutes of Health, Bethesda, MD). The regions of interest were drawn according to an atlas of the mouse brain.36 BOLD signal intensity values in each regions of interest were extracted and normalized to the time of baseline (expressed as a percent change from baseline). Statistical analysis was performed to compare percent changes in BOLD signal intensity and activated pixels between baseline and each time point after CFA and dynorphin A (1–17) injection.
RNA Preparation and Semiquantitative Analysis by Reverse Transcription–Polymerase Chain Reaction (PCR)
Total RNA obtained from the spinal cord of mice was extracted using the SV Total RNA Isolation System (Promega Co., Madison, WI). The lumbar spinal cord was quickly removed after mice were decapitated and homogenized in ice-cold lysis buffer containing β-mercaptoethanol following the manufacturer's instructions. First-strand complementary DNA was prepared as described,37 and the prodynorphin gene was amplified in 50 μ l of a PCR solution containing MgCl2, dNTP mix, and DNA polymerase with either synthesized primers (prodynorphin: 5′-GTG CAG TGA GGA TTC AGG ATG GG-3′[sense] and 5′-GAG CTT GGC TAG TGC ACT GTA GC-3′[antisense], glyceraldehyde-3-phosphate dehydrogenase: 5′-CCC ACG GCA AGT TCA ACG G-3′[sense] and 5′-CTT TCC AGA GGG GCC ATC CA-3′[antisense]). Samples were heated to 94° C for 2 min, 55° C for 2 min, and 72° C for 3 min and cycled 29 times through 94° C for 1 min, 55° C for 2 min, and 72° C for 3 min. The final incubation was at 72° C for 7 min. The mixture was subjected to 1% agarose gel for electrophoresis with the indicated markers and primers for the internal standard (glyceraldehyde-3-phosphate dehydrogenase). Each sample was applied to more than two lanes in the same gel. The agarose gel was stained with ethidium bromide and photographed with ultraviolet transillumination. The intensity of the bands was analyzed and quantified by computer-assisted densitometry using ImageJ (free download software developed by National Institutes of Health). For the control, the different intensities of each band obtained from mice treated with saline were analyzed, and the average intensity was calculated. Each control intensity was then compared again with the average intensity to calculate the standard error. Under these conditions, the intensities of bands for samples obtained from CFA-treated mice were analyzed and compared with the average intensity for mice treated with saline. Finally, the percent of control with standard error for each sample was quantified.
Quantitative Analysis by Real-time PCR
Fast SYBR Green Master Mix (2 ×; Applied Biosystems, Inc., Foster City, CA) was used as the basis for the reaction mixture in the real-time PCR assay. Each gene prepared by the above procedure was amplified in 20 μ l of a PCR solution containing 10 μ l of the Fast SYBR Green Master Mix (2 ×) with synthesized primers for PDYN (sense: 5′-TTT GGC AAC GGA AAA GAA TC-3′, antisense: 5′-CAT AGC GTT TGG CCT GTT TT-3′) or β-actin (sense: 5′-CAG CTT CTT TGC AGC TCC TT-3′, antisense: 5′-TCA CCC ACA TAG GAG TCC TT-3′). In addition to each sample, each test run included a no-target control that contained reaction mixture and PCR-grade water. PCR with a StepOnePlus (Applied Biosystems, Inc., Foster City, CA) was performed with the following cycling conditions: 95° C for 20 s, followed by cycled 45 cycles of 95° C for 3 s and 60° C for 30 s. Fluorescence detection was conducted after each extension step.
Spinal Cord Sample Preparation and Immunohistochemistry
Sample preparation and immunohistochemistry were performed following the methods previously described.38 Six hours after intraplantar injection, mice were deeply anesthetized with isoflurane and intracardially perfusion fixed with freshly prepared 4% paraformaldehyde in 0.1 m phosphate buffer saline (PBS), pH 7.4. After perfusion, the lumbar spinal cord was quickly removed and postfixed in 4% paraformaldehyde for 2 h and then permeated with 20% sucrose in 0.1 m PBS for 1 day and 30% sucrose in 0.1 m PBS for 2 days with agitation. The L5 lumbar spinal cord segments were then frozen in an embedding compound (Sakura Finetechnical, Tokyo, Japan) on isopentane using liquid nitrogen and stored at −30° C until use. Frozen spinal cord segments were cut with a freezing cryostat (Leica CM 1510; Leica, Wetzlar, Germany) at a thickness of 10 μ m and thaw mounted on poly-l-lysine-coated glass slides.
The spinal cord sections were blocked in 20% normal goat serum with 0.1% Triton X in 0.01 m PBS for 1 h at room temperature. The primary antibody [1:600 dynorphin A (1–17) (Phoenix Pharmaceuticals, Inc., Belmont, CA)] was diluted in 0.01 m PBS containing 20% normal goat serum with 0.1% Triton X and incubated for two nights at 4° C. The samples were then rinsed and incubated with an appropriate secondary antibody conjugated with Alexa 546 for 2 h at room temperature. Because the staining intensity might vary between experiments, control sections were included in each run of staining. The slides were then coverslipped with PermaFluor aqueous mounting medium (Immunon; Thermo Electron, Pittsburgh, PA). All sections were observed with a fluorescence microscope (Olympus BX-80; Olympus, Tokyo, Japan) and photographed with a digital camera (CoolSNAP HQ; Olympus).
Statistical Analysis
Dat a are expressed as the mean with SEM. One-and two-way ANOVAs with independent and repeated measures, as well as planned comparisons or Student t  tests, were used as appropriate for the experimental design. Multiple comparisons were performed using Dunnett or Bonferroni post hoc  test, where appropriate. All statistical analyses were performed with Prism version 5.0a (GraphPad Software, Inc., San Diego, CA).
Results
Pain-like Behaviors Induced by CFA Injection in Prodynorphin Knockout and Wild-type Mice
Genotyping of the offspring from prodynorphin knockout (−/−) mice was confirmed by PCR analysis using DNA extracted from the ear (data not shown). W e investigated whether lack of the prodynorphin gene could influence inflammatory pain using these genotype mice. In wild-type mice, CFA injection caused significant flinching or licking behaviors (fig. 1). These behaviors started just after CFA injection and lasted for more than 20 min. However, the numbers and durations of these behaviors were clearly decreased in prodynorphin knockout mice with CFA injection (fig. 1). Two-way ANOVA showed a significant interaction between genotype and treatment (flinching, F  (1,14) = 4.736, P  = 0.0471; licking, F  (1,14) = 6.699, P  = 0.0215; duration, F  (1,14) = 31.72, P  < 0.0001), a significant effect of treatment (flinching, F  (1,14) = 19.39, P  = 0.0006; licking, F  (1,14) = 17.53, P  = 0.0009; duration, F  (1,14) = 59.56, P  < 0.0001), and a significant effect of genotype (flinching, F  (1,14) = 5.291, P  = 0.0373; licking, F  (1,14) = 10.94, P  = 0.0053; duration, F  (1,14) = 43.78, P  < 0.0001). Post hoc  comparison indicated a significant difference between the saline in wild-type group and the CFA in wild-type group (flinching response: F  (3,12) = 24.87, P  < 0.0001; licking response: F  (3,12) = 70.84, P  < 0.0001; total duration of responses: F  (3,12) = 58.98; P  < 0.0001) and a significant difference between CFA in wild-type group and CFA in prodynorphin knockout group (flinching response: F  (3,12) = 24.87, P  = 0.0019; licking response: F  (3,12) = 70.84, P  < 0.0001; total duration of responses: F  (3,12) = 58.98, P  < 0.0001).
Fig. 1. Effect of an intraplantar injection of complete Freund's adjuvant (CFA) on spontaneous pain-like behaviors in wild-type (+/+) and prodynorphin (PDYN) knockout (−/−) mice. Immediately after intraplantar injection, the number of flinching (A-i  ) or licking (A-ii  ) behaviors was counted, and the total duration of flinching and licking behaviors was measured (B  ) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P  < 0.001, wild-type mouse with intraplantar injection of saline versus  wild-type mouse with intraplantar injection of CFA; $$ P  < 0.01, $$$ P  < 0.001, wild-type mouse with intraplantar injection of CFA versus  PDYN knockout mouse with intraplantar injection of CFA.
Fig. 1. Effect of an intraplantar injection of complete Freund's adjuvant (CFA) on spontaneous pain-like behaviors in wild-type (+/+) and prodynorphin (PDYN) knockout (−/−) mice. Immediately after intraplantar injection, the number of flinching (A-i 
	) or licking (A-ii 
	) behaviors was counted, and the total duration of flinching and licking behaviors was measured (B 
	) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P 
	< 0.001, wild-type mouse with intraplantar injection of saline versus 
	wild-type mouse with intraplantar injection of CFA; $$ P 
	< 0.01, $$$ P 
	< 0.001, wild-type mouse with intraplantar injection of CFA versus 
	PDYN knockout mouse with intraplantar injection of CFA.
Fig. 1. Effect of an intraplantar injection of complete Freund's adjuvant (CFA) on spontaneous pain-like behaviors in wild-type (+/+) and prodynorphin (PDYN) knockout (−/−) mice. Immediately after intraplantar injection, the number of flinching (A-i  ) or licking (A-ii  ) behaviors was counted, and the total duration of flinching and licking behaviors was measured (B  ) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P  < 0.001, wild-type mouse with intraplantar injection of saline versus  wild-type mouse with intraplantar injection of CFA; $$ P  < 0.01, $$$ P  < 0.001, wild-type mouse with intraplantar injection of CFA versus  PDYN knockout mouse with intraplantar injection of CFA.
×
Time Course of the Effect of Intraplantar Injection of CFA on BOLD Signal Intensity in Several Brain Regions in Wild-type and Prodynorphin Knockout Mice
Next, we investigated the changes in BOLD signal intensity in two brain regions after intraplantar injection of CFA. As shown in figures 2A–C, injection of CFA caused positive signal activity in the cingulate cortex (A-i), somatosensory cortex (B-i), and insular cortex (C-i) of wild-type mice compared with prodynorphin knockout mice. In the cingulate cortex, injection of CFA produced a n increase in signal intensity (fig. 2A-ii). In the somatosensory cortex of wild-type mice, injection of CFA also produced an increase in signal intensity after injection (fig. 2B-ii). In the insular cortex of wild-type mice, similar to somatosensory area, injection of CFA produced a n increase in signal intensity after injection (fig. 2C-ii). These effects were not seen in mice that lacked the prodynorphin gene. Statistical analyses were done with two-way ANOVA followed by Bonferroni test (cingulate cortex: interaction between genotype and time: F  (7,48) = 1.138, P  = 0.356; effect of genotype, F  (1,48) = 14.28, P  = 0.0004; effect of time, F  (7,48) = 1.348, P  = 0.2491; somatosensory cortex: interaction between genotype and time: F  (7,48) = 0.996, P  = 0.446; effect of genotype, F  (1,48) = 27.77, P  < 0.0001; effect of time, F  (7,48) = 0.984, P  = 0.4543; insular cortex: interaction between genotype and time: F  (7,48) = 4.799, P  = 0.0004; effect of genotype, F  (1,48) = 151.3, P  < 0.0001; effect of time, F  (7,48) = 2.933, P  = 0.0122) (figs. 2A-ii–C-ii). As shown in figures 2D and E, injection of CFA caused positive signal activity in the medial thalamic nuclei (D-i) and the lateral thalamic nuclei (E-i) of wild-type mice compared with prodynorphin knockout mice. The medial thalamic nuclei and lateral thalamic nuclei were also activated by CFA injection in wild-type mice but not in prodynorphin knockout mice. Statistical analyses were done with two-way ANOVA followed by Bonferroni test (medial thalamic nuclei: interaction between genotype and time: F  (7,48) = 0.730, P  = 0.6475; effect of genotype, F  (1,48) = 14.62, P  = 0.0004; effect of time, F  (7,48) = 0.856, P  = 0.5476; lateral thalamic nuclei: interaction between genotype and time: F  (7,48) = 0.501, P  = 0.8289; effect of genotype, F  (1,48) = 11.72, P  = 0.0013; effect of time, F  (7,48) = 0.563, P  = 0.7821) (figs. 2D-ii and E-ii). Under these conditions, control mice of both genotypes that had been injected with saline failed to show activation in any brain regions (data not shown).
Fig. 2. Time course of the effect of intraplantar injection of complete Freund's adjuvant (CFA) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial halamic region (mTH), and lateral thalamic region (lTH) in wild-type (WT) and prodynorphin (PDYN) knockout (KO) mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 6 h (A-i  : CG, B-i  : S1, C-i  : IC, D-i  ; mTH, E-i  ; lTH) after intraplantar injection of CFA in WT and KO mice. Intraplantar injection of CFA produced a significant increase in BOLD signal in the CG (A-ii  ), S1 (B-ii  ), IC (C-ii  ), mTH (D-ii  ), and lTH (E-ii  ) of wild-type mice. Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice.
Fig. 2. Time course of the effect of intraplantar injection of complete Freund's adjuvant (CFA) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial halamic region (mTH), and lateral thalamic region (lTH) in wild-type (WT) and prodynorphin (PDYN) knockout (KO) mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 6 h (A-i 
	: CG, B-i 
	: S1, C-i 
	: IC, D-i 
	; mTH, E-i 
	; lTH) after intraplantar injection of CFA in WT and KO mice. Intraplantar injection of CFA produced a significant increase in BOLD signal in the CG (A-ii 
	), S1 (B-ii 
	), IC (C-ii 
	), mTH (D-ii 
	), and lTH (E-ii 
	) of wild-type mice. Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice.
Fig. 2. Time course of the effect of intraplantar injection of complete Freund's adjuvant (CFA) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial halamic region (mTH), and lateral thalamic region (lTH) in wild-type (WT) and prodynorphin (PDYN) knockout (KO) mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 6 h (A-i  : CG, B-i  : S1, C-i  : IC, D-i  ; mTH, E-i  ; lTH) after intraplantar injection of CFA in WT and KO mice. Intraplantar injection of CFA produced a significant increase in BOLD signal in the CG (A-ii  ), S1 (B-ii  ), IC (C-ii  ), mTH (D-ii  ), and lTH (E-ii  ) of wild-type mice. Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice.
×
Effects of Intrathecal Injection of Antiserum to Dynorphin A (1–17) on the Response Induced by CFA Injection
To confirm the possible involvement of spinal dynorphin A (1–17) in the pain-related response induced by CFA injection, we performed intrathecal injection of antiserum against dynorphin A (1–17) and evaluated its effect on CFA-mediated pain-related responses. Dynorphin A (1–17) antiserum significantly decreased the number of and shortened the duration of flinching or licking responses compared with those in control serum-treated mice (fig. 3). The two-way ANOVA indicated a significant interaction between pretreatment and posttreatment (flinching, F  (1,23) = 48.89, P  < 0.0001; licking, F  (1,23) = 14.83, P  = 0.0008; duration, F  (1,23) = 14.19, P  = 0.001), a significant effect of pretreatment (flinching, F  (1,23) = 122.8, P  < 0.0001; licking, F  (1,23) = 42.02, P  < 0.0001; duration, F  (1,23) = 22.43, P  < 0.0001), and a significant effect of posttreatment (flinching, F  (1,23) = 56.6, P  < 0.0001; licking, F  (1,23) = 12.03, P  = 0.0021; duration, F  (1,23) = 8.825, P  = 0.0068). Post hoc  comparison indicated a significant difference between the control serum-saline group and control serum-CFA group (flinching response: F  (3,23) = 76.95, P  < 0.0001; licking response: F  (3,23) = 23.04, P  < 0.0001; total duration of responses: F  (3,23) = 15.38; P  < 0.0001) and a significant difference between control serum-CFA group and dynorphin A (1–17) antiserum-CFA group (flinching response: F  (3,23) = 76.95, P  < 0.0001; licking response: F  (3,23) = 23.04, P  < 0.0001; total duration of responses: F  (3,23) = 23.04, P  = 0.00025).
Fig. 3. Effect of intrathecal pretreatment with antiserum against dynorphin A (1–17) on pain-like behaviors evoked by intraplantar injection of complete Freund's adjuvant (CFA). Groups of mice were treated intrathecal with antiserum against dynorphin A (1–17) (Dynorphin A/S; 1:100) or control serum 30 min before the intraplantar injection of CFA. Immediately after the intraplantar injection of saline or CFA, the number of flinching (A-i  ) or licking (A-ii  ) behaviors was counted, and the duration of flinching and licking behaviors was measured (B  ) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P  < 0.001, control serum-saline group versus  control serum-CFA group; $$$ P  < 0.001, control serum-CFA group versus  Dynorphin A/S-CFA group.
Fig. 3. Effect of intrathecal pretreatment with antiserum against dynorphin A (1–17) on pain-like behaviors evoked by intraplantar injection of complete Freund's adjuvant (CFA). Groups of mice were treated intrathecal with antiserum against dynorphin A (1–17) (Dynorphin A/S; 1:100) or control serum 30 min before the intraplantar injection of CFA. Immediately after the intraplantar injection of saline or CFA, the number of flinching (A-i 
	) or licking (A-ii 
	) behaviors was counted, and the duration of flinching and licking behaviors was measured (B 
	) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P 
	< 0.001, control serum-saline group versus 
	control serum-CFA group; $$$ P 
	< 0.001, control serum-CFA group versus 
	Dynorphin A/S-CFA group.
Fig. 3. Effect of intrathecal pretreatment with antiserum against dynorphin A (1–17) on pain-like behaviors evoked by intraplantar injection of complete Freund's adjuvant (CFA). Groups of mice were treated intrathecal with antiserum against dynorphin A (1–17) (Dynorphin A/S; 1:100) or control serum 30 min before the intraplantar injection of CFA. Immediately after the intraplantar injection of saline or CFA, the number of flinching (A-i  ) or licking (A-ii  ) behaviors was counted, and the duration of flinching and licking behaviors was measured (B  ) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P  < 0.001, control serum-saline group versus  control serum-CFA group; $$$ P  < 0.001, control serum-CFA group versus  Dynorphin A/S-CFA group.
×
Increase in the Expression of Spinal Prodynorphin Messenger RNA at the Early Phase of Inflammatory Pain after Intraplantar Injection of CFA in Mice
In the reverse transcription–PCR assay, the expression of prodynorphin messenger RNA (mRNA) on the ipsilateral side of the spinal cord obtained from CFA-treated mice was significantly increased at 1, 3, and 6 h after CFA injection compared with that in saline-treated mice (1 h: P  = 0.014, 3 h: P  = 0.0002, 6 h: P  = 0.0008 vs.  saline-treated group; figs. 4A and B). In the contralateral spinal cord obtained from CFA-treated mice, there were no significant differences in the expression level of prodynorphin mRNA compared with those on the contralateral side of saline-treated mice (data not shown). In agreement with the results of reverse transcription–PCR, we confirmed that the expression of PDYN mRNA was significantly increased in the spinal cord of mice with CFA treatment compared with that in saline-treated mice at 6 h after CFA injection using real-time PCR (P  = 0.042 vs.  saline-treated group; fig. 4C).
Fig. 4. (A  ) Representative reverse transcription–polymerase chain reaction for prodynorphin (PDYN) messenger RNAs on the ipsilateral side of spinal cords obtained from saline-or complete Freund's adjuvant (CFA)-injected mice. Spinal cord samples were prepared at 1, 3, and 6 h after saline or CFA injection (B  ). The intensity of the bands was determined semiquantitatively using ImageJ (National Institutes of Health). The values for PDYN messenger RNA were normalized by the value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA. The value in CFA-injected mice is expressed as a percentage of the increase in saline-injected mice. Each column represents the mean ± SEM of three samples. Student t  test: **P  < 0.01, ***P  < 0.001, versus  saline group. (C  ) Quantitative analysis of PDYN messenger RNA on the spinal cord of saline-or CFA-treated mice at 6 h after the injection. (C-i  ) Amplification plots of fluorescence intensities versus  PCR cycle numbers in each sample are displayed. (C-ii  ) Each column represents the mean ± SEM of three samples. *P  < 0.05 versus  saline-treated group. (D  ) Change in dynorphin A (1–17)-like immunoreactivity on the superficial laminae of the ipsilateral dorsal horn after CFA injection. Photomicrographs show immunofluorescent staining of dynorphin A (1–17) on the superficial layers of the ipsilateral spinal dorsal horn at 6 h after saline (D-i  ) or CFA injection. Dynorphin A-like immunoreactivity observed on the superficial laminae of the ipsilateral dorsal horn after CFA injection (D-ii  ) was significantly increased compared with that after saline injection (D-i  ). Scale bars = 50 μ m.
Fig. 4. (A 
	) Representative reverse transcription–polymerase chain reaction for prodynorphin (PDYN) messenger RNAs on the ipsilateral side of spinal cords obtained from saline-or complete Freund's adjuvant (CFA)-injected mice. Spinal cord samples were prepared at 1, 3, and 6 h after saline or CFA injection (B 
	). The intensity of the bands was determined semiquantitatively using ImageJ (National Institutes of Health). The values for PDYN messenger RNA were normalized by the value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA. The value in CFA-injected mice is expressed as a percentage of the increase in saline-injected mice. Each column represents the mean ± SEM of three samples. Student t 
	test: **P 
	< 0.01, ***P 
	< 0.001, versus 
	saline group. (C 
	) Quantitative analysis of PDYN messenger RNA on the spinal cord of saline-or CFA-treated mice at 6 h after the injection. (C-i 
	) Amplification plots of fluorescence intensities versus 
	PCR cycle numbers in each sample are displayed. (C-ii 
	) Each column represents the mean ± SEM of three samples. *P 
	< 0.05 versus 
	saline-treated group. (D 
	) Change in dynorphin A (1–17)-like immunoreactivity on the superficial laminae of the ipsilateral dorsal horn after CFA injection. Photomicrographs show immunofluorescent staining of dynorphin A (1–17) on the superficial layers of the ipsilateral spinal dorsal horn at 6 h after saline (D-i 
	) or CFA injection. Dynorphin A-like immunoreactivity observed on the superficial laminae of the ipsilateral dorsal horn after CFA injection (D-ii 
	) was significantly increased compared with that after saline injection (D-i 
	). Scale bars = 50 μ m.
Fig. 4. (A  ) Representative reverse transcription–polymerase chain reaction for prodynorphin (PDYN) messenger RNAs on the ipsilateral side of spinal cords obtained from saline-or complete Freund's adjuvant (CFA)-injected mice. Spinal cord samples were prepared at 1, 3, and 6 h after saline or CFA injection (B  ). The intensity of the bands was determined semiquantitatively using ImageJ (National Institutes of Health). The values for PDYN messenger RNA were normalized by the value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA. The value in CFA-injected mice is expressed as a percentage of the increase in saline-injected mice. Each column represents the mean ± SEM of three samples. Student t  test: **P  < 0.01, ***P  < 0.001, versus  saline group. (C  ) Quantitative analysis of PDYN messenger RNA on the spinal cord of saline-or CFA-treated mice at 6 h after the injection. (C-i  ) Amplification plots of fluorescence intensities versus  PCR cycle numbers in each sample are displayed. (C-ii  ) Each column represents the mean ± SEM of three samples. *P  < 0.05 versus  saline-treated group. (D  ) Change in dynorphin A (1–17)-like immunoreactivity on the superficial laminae of the ipsilateral dorsal horn after CFA injection. Photomicrographs show immunofluorescent staining of dynorphin A (1–17) on the superficial layers of the ipsilateral spinal dorsal horn at 6 h after saline (D-i  ) or CFA injection. Dynorphin A-like immunoreactivity observed on the superficial laminae of the ipsilateral dorsal horn after CFA injection (D-ii  ) was significantly increased compared with that after saline injection (D-i  ). Scale bars = 50 μ m.
×
Changes in Dynorphin A (1–17)–like Immunoreactivity after Intraplantar Injection of CFA in the Superficial Dorsal Horn of the Mouse Spinal Cord
To investigate a possible change in the level of dynorphin A (1–17) in the superficial dorsal horn of CFA-treated mice, immunohistochemical studies were performed. Dynorphin A (1–17)-like immunoreactivity was detected on the superficial laminae of the ipsilateral side of the L5 lumbar spinal dorsal horn with saline treatment (fig. 4D-i). Six hours after CFA injection, dynorphin A (1–17)-like immunoreactivity on the superficial laminae of the ipsilateral side of the L5 lumbar spinal dorsal horn was markedly increased compared with that with saline injection (fig. 4D-ii).
Pain-like Behaviors Induced by Intrathecal Injection of Dynorphin A (1–17) in Mice
Next, we observed pain-like behaviors induced by intrathecal dynorphin A (1–17) in mice. As shown in figure 5, intrathecal injection of dynorphin A (1–17) produced a significant increase in both the number of biting or licking responses and the duration of these responses in a dose-dependent manner. Statistical analyses were done with one-way ANOVA followed by Dunnett test (biting or licking responses: F  (3,17) = 6.084, 0.3 pmol: P  = 0.6584, 3 pmol: P  = 0.039, 30 pmol: P  = 0.0033; total duration of responses: F  (3,17) = 10.94, 0.3 pmol: P  = 0.2108, 3 pmol: P  = 0.0018, 30 pmol: P  = 0.0002; vs.  intrathecal vehicle group; figs. 5A and B). These responses started a pproximately 4 or 5 min after intrathecal injection and lasted for more than 20 min. The control group-injected intrathecal with vehicle did not exhibit these behaviors.
Fig. 5. Effect of a single intrathecal injection of dynorphin A (1–17) on pain-like behavior. Groups of mice were treated intrathecal injection with dynorphin A (1–17) (0.3, 3, 30 pmol) or saline. Immediately after intrathecal injection, the number of licking or biting behaviors was counted (A  ), and the duration of licking or biting behaviors was measured (B  ) for 20 min after the intrathecal injection of dynorphin A (1–17). The intrathecal injection of dynorphin A (1–17) produced a dose-dependent increase in pain-like behaviors compared with control mice injected intrathecal with vehicle. Each point indicates the mean ± SEM of four to six mice. Dunnett test: *P  < 0.05, **P  < 0.01, ***P  < 0.001, versus  intrathecal vehicle group.
Fig. 5. Effect of a single intrathecal injection of dynorphin A (1–17) on pain-like behavior. Groups of mice were treated intrathecal injection with dynorphin A (1–17) (0.3, 3, 30 pmol) or saline. Immediately after intrathecal injection, the number of licking or biting behaviors was counted (A 
	), and the duration of licking or biting behaviors was measured (B 
	) for 20 min after the intrathecal injection of dynorphin A (1–17). The intrathecal injection of dynorphin A (1–17) produced a dose-dependent increase in pain-like behaviors compared with control mice injected intrathecal with vehicle. Each point indicates the mean ± SEM of four to six mice. Dunnett test: *P 
	< 0.05, **P 
	< 0.01, ***P 
	< 0.001, versus 
	intrathecal vehicle group.
Fig. 5. Effect of a single intrathecal injection of dynorphin A (1–17) on pain-like behavior. Groups of mice were treated intrathecal injection with dynorphin A (1–17) (0.3, 3, 30 pmol) or saline. Immediately after intrathecal injection, the number of licking or biting behaviors was counted (A  ), and the duration of licking or biting behaviors was measured (B  ) for 20 min after the intrathecal injection of dynorphin A (1–17). The intrathecal injection of dynorphin A (1–17) produced a dose-dependent increase in pain-like behaviors compared with control mice injected intrathecal with vehicle. Each point indicates the mean ± SEM of four to six mice. Dunnett test: *P  < 0.05, **P  < 0.01, ***P  < 0.001, versus  intrathecal vehicle group.
×
Time Course of the Effect of Intrathecal Injection of Dynorphin A (1–17) on BOLD Signal Intensity in Different Brain Regions in Mice
We also investigated the changes in BOLD signal intensity in different brain regions after the direct injection of dynorphin A (1–17) in the spinal cord of mice. Intrathecal injection of 30-pmol dynorphin A (1–17), which induced maxim u m pain-related responses in the behavior study, caused a significant increase in BOLD signal intensity in the cingulate cortex, somatosensory cortex, and insular cortex of mice compared with the basal activity. Although each of the signal intensities was increased compared with the basal intensity, there were slight differences in the time course of the increase between these regions. The cingulate cortex was significantly activated after intrathecal injection of dynorphin A (1–17) (fig. 6A). In the somatosensory cortex, intrathecal injection of dynorphin A (1–17) produced a significant increase in signal intensity (fig. 6B). In the insular cortex, the activation appeared after intrathecal injection of dynorphin A (1–17) (fig. 6C). In the medial and lateral thalamic nuclei, both BOLD signal intensities were significantly changed after intrathecal injection of dynorphin A (1–17) (figs. 6D and E). These activations reverted to their basal levels at 6 h after dynorphin A (1–17) injection. Control mice that had been injected intrathecal with saline did not show such activation in these regions (fig. 6). Statistical analys e s were done with two-way ANOVA followed by Bonferroni test (cingulate cortex: interaction between treatme n t and time: F  (7,64) = 0.965, P  = 0.4642; effect of treatment, F  (1,64) = 19.70, P  < 0.0001; effect of time, F  (7,64) = 0.938, P  = 0.4837; somatosensory cortex: interaction between treatment and time: F  (7,64) = 5.413, P  < 0.0001; effect of treatment, F  (1,64) = 19.85, P  < 0.0001; effect of time, F  (7,64) = 6.933, P  < 0.0001; insular cortex: interaction between treatment and time: F  (7,64) = 2.474, P  < 0.026; effect of treatment, F  (1,64) = 15.79, P  = 0.0002; effect of time, F  (7,64) = 2.977, P  = 0.0091; medial thalamic nuclei: interaction between treatment and time: F  (7,64) = 1.655, P  = 0.1362; effect of treatment, F  (1,64) = 28.97, P  < 0.0001; effect of time, F  (7,64) = 2.242, P  = 0.0419; lateral thalamic nuclei: interaction between treatment and time: F  (7,64) = 1.500, P  = 0.1834; effect of treatment, F  (1,64) = 12.78, P  = 0.0007; effect of time, F  (7,64) = 1.581, P  = 0.1571).
Fig. 6. Time course of the effects of intrathecal injection of dynorphin A (1–17) (30 pmol) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial thalamic region (mTH), and lateral thalamic region (lTH) in mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 2 h (A-i  : CG), 0 or 1 h (B-i  : S1, C-i  : IC, D-i  ; mTH), and 0 or 0.5 h (E-i  : lTH) after the intrathecal injection of dynorphin A (1–17) in mice. The intrathecal injection of dynorphin A (1–17) produced a significant increase in BOLD signal compared with basal intensity (A-ii  : CG; B-ii  : S1; C-ii  : IC; D-ii  mTH; E-ii  : lTH). Each point indicates the mean ± SEM for five mice. CFA = complete Freund's adjuvant.
Fig. 6. Time course of the effects of intrathecal injection of dynorphin A (1–17) (30 pmol) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial thalamic region (mTH), and lateral thalamic region (lTH) in mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 2 h (A-i 
	: CG), 0 or 1 h (B-i 
	: S1, C-i 
	: IC, D-i 
	; mTH), and 0 or 0.5 h (E-i 
	: lTH) after the intrathecal injection of dynorphin A (1–17) in mice. The intrathecal injection of dynorphin A (1–17) produced a significant increase in BOLD signal compared with basal intensity (A-ii 
	: CG; B-ii 
	: S1; C-ii 
	: IC; D-ii 
	mTH; E-ii 
	: lTH). Each point indicates the mean ± SEM for five mice. CFA = complete Freund's adjuvant.
Fig. 6. Time course of the effects of intrathecal injection of dynorphin A (1–17) (30 pmol) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial thalamic region (mTH), and lateral thalamic region (lTH) in mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 2 h (A-i  : CG), 0 or 1 h (B-i  : S1, C-i  : IC, D-i  ; mTH), and 0 or 0.5 h (E-i  : lTH) after the intrathecal injection of dynorphin A (1–17) in mice. The intrathecal injection of dynorphin A (1–17) produced a significant increase in BOLD signal compared with basal intensity (A-ii  : CG; B-ii  : S1; C-ii  : IC; D-ii  mTH; E-ii  : lTH). Each point indicates the mean ± SEM for five mice. CFA = complete Freund's adjuvant.
×
Effect of Pretreatment with MK-801 on Intrathecal Dynorphin A (1–17)–induced Pain-like Behaviors
To determine the possible involvement of spinal NMDA receptors in dynorphin A (1–17)-induced pain-like behaviors, we observed the influence of intrathecal pretreatment with the NMDA receptor antagonist, MK-801. As shown in figure 7, MK-801 pretreatment produced a dose-dependent inhibition of pain-like responses that could be caused by after intrathecal dynorphin A (1–17) injection. Statistical analys e s were done with one-way ANOVA followed by Dunnett test (biting or licking responses: F  (2,14) = 15.65, 0.3 nmol: P  = 0.0022, 1 nmol: P  = 0.0002; total duration of responses: F  (2,14) = 13.28; 0.3 nmol: P  = 0.0052, 1 nmol: P  = 0.0003; vs.  vehicle–dynorphin A (1–17)-treated group). Under these conditions, control mice of both, that is, pretreatment with saline and MK-801 that had been injected with saline, failed to show these behaviors (data not shown).
Fig. 7. Effect of intrathecal pretreatment with MK-801 on dynorphin A (1–17)-mediated pain-like response. Groups of mice were treated intrathecal with a noncompetitive N  -methyl-d-a spartic acid receptor antagonist, MK-801 (1 nmol/mouse), or vehicle 30 min before the intrathecal injection of dynorphin A (1–17) (30 pmol). Immediately after the intrathecal injection of dynorphin A (1–17), the number of licking or biting behaviors was counted (A  ), and the duration of licking or biting behaviors was measured (B  ) for 20 min after the intrathecal injection of dynorphin A (1–17). Dunnett test: $$ P  < 0.01, $$$ P  < 0.001, versus  saline-dynorphin A (1–17) group.
Fig. 7. Effect of intrathecal pretreatment with MK-801 on dynorphin A (1–17)-mediated pain-like response. Groups of mice were treated intrathecal with a noncompetitive N 
	-methyl-d-a spartic acid receptor antagonist, MK-801 (1 nmol/mouse), or vehicle 30 min before the intrathecal injection of dynorphin A (1–17) (30 pmol). Immediately after the intrathecal injection of dynorphin A (1–17), the number of licking or biting behaviors was counted (A 
	), and the duration of licking or biting behaviors was measured (B 
	) for 20 min after the intrathecal injection of dynorphin A (1–17). Dunnett test: $$ P 
	< 0.01, $$$ P 
	< 0.001, versus 
	saline-dynorphin A (1–17) group.
Fig. 7. Effect of intrathecal pretreatment with MK-801 on dynorphin A (1–17)-mediated pain-like response. Groups of mice were treated intrathecal with a noncompetitive N  -methyl-d-a spartic acid receptor antagonist, MK-801 (1 nmol/mouse), or vehicle 30 min before the intrathecal injection of dynorphin A (1–17) (30 pmol). Immediately after the intrathecal injection of dynorphin A (1–17), the number of licking or biting behaviors was counted (A  ), and the duration of licking or biting behaviors was measured (B  ) for 20 min after the intrathecal injection of dynorphin A (1–17). Dunnett test: $$ P  < 0.01, $$$ P  < 0.001, versus  saline-dynorphin A (1–17) group.
×
Effect of Pretreatment with MK-801 on the Increase in BOLD Signal Intensity Induced by Intrathecal Injection of Dynorphin A (1–17)
Because the cingulate cortex, somatosensory cortex, insular cortex, and the medial and lateral thalamic nuclei were all activated by intrathecal injection of dynorphin A (1–17) in a time-dependent manner, we further investigated whether the activation of these brain regions could be changed by pretreatment with MK-801. After intrathecal injection of MK-801 (1 nmol) and before dynorphin A (1–17) administration, we compared the changes in the increased levels of signal intensity in brain regions after intrathecal injection of dynorphin A (1–17), when the most significant increase in BOLD signal intensity was observed. As for the cortex regions, the increased levels of BOLD intensity at 2 h (in the cingulate cortex) or 1 h (in somatosensory cortex and the insular cortex) induced by intrathecal dynorphin A (1–17) were significantly suppressed by pretreatment with MK-801 (figs. 8A–C). In the medial and lateral thalamic nuclei, increased levels of BOLD intensity induced by dynorphin A (1–17) were also blocked by pretreatment with MK-801 (figs. 8D and E). Two-way ANOVA showed a significant interaction between phase and pretreatment (cingulate cortex, F  (1,16) = 6.517, P  = 0.0213; somatosensory cortex, F  (1,16) = 13.06, P  = 0.0023; insular cortex, F  (1,16) = 26.31, P  = 0.0001; medial thalamic nuclei, F  (1,16) = 9.884, P  = 0.0063; lateral thalamic nuclei, F  (1,16) = 8.402, P  = 0.0105), a significant effect of phase (cingulate cortex, F  (1,16) = 10.17, P  = 0.0057; somatosensory cortex, F  (1,16) = 29.52, P  < 0.0001; insular cortex, F  (1,16) = 34.81, P  < 0.0001; medial thalamic nuclei, F  (1,16) = 15.30, P  = 0.0012; lateral thalamic nuclei, F  (1,16) = 6.496, P  = 0.0215), and a significant effect of pretreatment (cingulate cortex, F  (1,16) = 6.427, P  = 0.0221; somatosensory cortex, F  (1,16) = 13.35, P  = 0.0021; insular cortex, F  (1,16) = 25.20, P  = 0.0001; medial thalamic nuclei, F  (1,16) = 11.64, P  = 0.0036; lateral thalamic nuclei, F  (1,16) = 9.030, P  = 0.0084). Post hoc  comparison indicated a significant difference between the basal intensity of saline-pretreated groups versus  dynorphin challenge in saline-pretreated groups (cingulate cortex, F  (3,16) = 7.703, P  = 0.0048; somatosensory cortex, F  (3,16) = 18.64, P  < 0.0001; insular cortex, F  (3,16) = 28.77, P  < 0.0001; medial thalamic nuclei, F  (3,16) = 12.27, P  = 0.0008; lateral thalamic nuclei, F  (3,16) = 7.976, P  = 0.0085) and a significant difference between the dynorphin challenge in saline-pretreated groups versus  dynorphin challenge in MK-801-pretreated groups (cingulate cortex, F  (3,16) = 7.703, P  = 0.0144; somatosensory cortex, F  (3,16) = 18.64, P  < 0.0001; insular cortex, F  (3,16) = 28.77, P  < 0.0001; medial thalamic nuclei, F  (3,16) = 12.27, P  = 0.00165; lateral thalamic nuclei, F  (3,16) = 7.976, P  = 0.0043).
Fig. 8. The effect of the pretreatment of the MK-801 on increase in dynorphin-induced signal intensity (A  : cingulate cortex (CG); B  : somatosensory cortex (S1); C  : insula cortex (IC); D  : medial thalamic region (mTH); E  : lateral thalamic region (lTH)). Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice. Bonferroni test: **P  < 0.01, ***P  < 0.001, the basal intensity of saline-pretreated groups versus  dynorphin challenge in saline-pretreated groups; $ P  < 0.05, $$ P  < 0.01, $$$ P  < 0.001, the dynorphin challenge in saline-pretreated groups versus  dynorphin challenge in MK-801-pretreated groups.
Fig. 8. The effect of the pretreatment of the MK-801 on increase in dynorphin-induced signal intensity (A 
	: cingulate cortex (CG); B 
	: somatosensory cortex (S1); C 
	: insula cortex (IC); D 
	: medial thalamic region (mTH); E 
	: lateral thalamic region (lTH)). Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice. Bonferroni test: **P 
	< 0.01, ***P 
	< 0.001, the basal intensity of saline-pretreated groups versus 
	dynorphin challenge in saline-pretreated groups; $ P 
	< 0.05, $$ P 
	< 0.01, $$$ P 
	< 0.001, the dynorphin challenge in saline-pretreated groups versus 
	dynorphin challenge in MK-801-pretreated groups.
Fig. 8. The effect of the pretreatment of the MK-801 on increase in dynorphin-induced signal intensity (A  : cingulate cortex (CG); B  : somatosensory cortex (S1); C  : insula cortex (IC); D  : medial thalamic region (mTH); E  : lateral thalamic region (lTH)). Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice. Bonferroni test: **P  < 0.01, ***P  < 0.001, the basal intensity of saline-pretreated groups versus  dynorphin challenge in saline-pretreated groups; $ P  < 0.05, $$ P  < 0.01, $$$ P  < 0.001, the dynorphin challenge in saline-pretreated groups versus  dynorphin challenge in MK-801-pretreated groups.
×
Discussion
Many studies have confirmed that endogenous opioid neuropeptides are associated with nociceptive transmission. In addition to endogenous opioid peptides, dynorphin A has been implicated in both pronociceptive and antinociceptive actions and is widely expressed throughout the central nervous system.3 Several studies have provided evidence that the nociceptive action of spinal dynorphin plays a role in the long-term maintenance of chronic pain, such as inflammatory pain8,21 and neuropathic pain.39 However, little, if any, is known about the role of dynorphin A in the acute phase of inflammatory pain. Therefore, in this study, we first investigated whether dynorphin A could be involved in pain-associated behaviors and brain activity in the acute phase of the peripheral inflammatory pain-like state using prodynorphin gene knockout mice and their wild-type mice.
Role of Dynorphin A in the Acute Phase of Inflammatory Pain
Prodynorphin gene knockout mice were indistinguishable from wild-type mice in terms of general behavior and development. Intraplantar injection of CFA increased the duration and number of spontaneous pain-associated behaviors such as flinching or licking in wild-type mice but not in prodynorphin gene knockout mice. Under the present condition, phMRI showed that intraplantar injection of CFA caused robust positive signal activity in the cingulate cortex, somatosensory cortex, insular cortex, and medial and lateral thalamic nuclei of wild-type mice. In contrast, these effects were diminished in prodynorphin knockout mice. Because cortical areas are activated by the receipt of noxious information from the spinothalamic tract, many neuroimaging studies have shown activity by demonstrating brain circuitry.40–42 These cortical representations of pain are called the pain matrix, including the somatosensory cortex, insular cortex, cingulate cortex, and prefrontal cortex.43 Among these areas, the cingulate cortex area is an affective-motivational component of pain and mainly receives information from the medial system of the spinothalamic tract.44,45 On the other hand, the somatosensory cortex area is a sensory-discriminative component of pain and mainly receives information from the lateral system of the spinothalamic tract. The insular cortex is basically considered to be not only in charge of the sensory-integrative component via  the lateral system but also involved in limbic integration by virtue of the medial system.43 The medial and lateral thalamic nuclei are also categorized as centers for pain perception, which relay sensory information to those cortical areas. Taken together, the present findings suggest that dynorphin A regulates spontaneous pain-associated behaviors and may play a crucial role in the activation of these pain-related brain regions in the acute phase of inflammatory pain.
Evidence for a Role of Spinal Dynorphin A
In the spinal cord, dynorphin A is principally localized in laminae 1 and 2, and in an inflammatory state it spreads to laminae 3 and 4 and deeper laminae.20,46 Furthermore, it has been reported that spinal dynorphin A immunoreactivity is increased in CFA-injected mice, 20,46 and the upregulation of spinal preprodynorphin mRNA is induced by peripheral inflammation.6,8,21,47,48 It was reported that in rats, incisional surgery increased the spinal cord dynorphin expression but did not drive microglial prostaglandin production or mechanical hypersensitivity.49 Although further investigation is required, these findings suggest that the involvement of spinal dynorphin in the different types of pain might be, in some cases, the species difference. In this study, the level of prodynorphin mRNA in the lumbar spinal cord of mice was increased at 1, 3, and 6 h after CFA injection. Furthermore, 6 h after intraplantar injection, CFA produced a marked increase in dynorphin A (1–17)-like immunoreactivity on the superficial layers of the ipsilateral side of the spinal cord. In this study, we demonstrated that CFA-induced pain-associated behaviors were suppressed by intrathecal injection of a specific antiserum for dynorphin A (1–17). These findings suggest that peripheral inflammation may release dynorphin A (1–17) with an increase in the expression of prodynorphin in the spinal cord, resulting in the induction of an early phase of an inflammatory pain-like state.
Multiple Mechanism of Spinal Dynorphin A
Prodynorphin transcription is regulated by several factors in a tissue-specific manner.50 It has been proposed that dynorphin expression is determined by a balance between gene transactivation and repression.51 In the spinal cord, the binding of transactivators including cyclic adenosine monophosphate response element-binding protein to cyclic adenosine monophosphate response element,52 the binding of c-fos  with c-jun  to an AP-1 element in the dynorphin promoter,53 and the derepression of DREAM54 play important roles in prodynorphin gene transcription. Several studies have reported that dynorphin A can affect several ion channels or nociceptive receptors through nonopioidergic mechanisms.3,15,18,19,55 In addition, those mechanisms involve the theory that dynorphin A may play a role in both antinociceptive and pronociceptive effects depending on its concentration: the former is due to endogenous κ-opioidergic action and the latter is due to glutaminergic actions through NMDA receptors.3 At physiologically lower concentrations, dynorphin A reduces calcium influx via  κ-opioid receptor activity, whereas at pathologically higher concentrations, dynorphin A causes increased intracellular calcium levels by activating NMDA receptors.15 Although further experiment is required for the measurement of released dynorphin A in the present case after CFA injection, we propose here that the noxious stimuli after CFA injection into the hind paw may consistently release high concentrations of dynorphin A in the spinal cord, leading to excitatory neurotransmission.
Involvement of NMDA Receptors in Spinal Dynorphin A–dependent Ascending Pain Transmission
It is well known that NMDA receptors initiate increases in the excitability of spinal neurons and cause the central sensitization of pain. Furthermore, a previous study suggested that NMDA receptors in the spinal cord play a major role in the increased BOLD signal in the somatosensory cortex by way of the spinothalamic pathway.56 Therefore, a subsequent study was undertaken to investigate whether NMDA receptors could be involved in spontaneous pain-associated behaviors or changes in brain activation induced by the intrathecal injection of dynorphin A. A single intrathecal injection of dynorphin A (1–17) caused an increase in pain-associated behaviors, and this effect was suppressed by pretreatment with MK-801. Under these conditions, we found here for the first time that dynorphin A produced a significant increase in signal intensity in the cingulate cortex, somatosensory cortex, insular cortex, and medial and lateral thalamic nuclei at 0.5–2 h after injection compared with the basal intensity, and this effect was abolished by pretreatment with MK-801. If we consider these findings, the present data suggest that an increase in spinal dynorphin A (1–17) may activate the transmission of ascending nociceptive information in the central nervous system via  NMDA receptors. Although additional research is needed to understand the mechanism of the activation of dynorphin A in the early phase of inflammatory pain, it seems likely that increased spinal dynorphin A may contribute not only to signals of acute pain but also to induction of chronic pain via  spinal NMDA receptors.
In view of the clinical application of the present information, we propose that spinal dynorphin may be an unique biomarker for pain.
Conclusion
The present data constitute novel evidence that loss of the prodynorphin gene prevents the ascending transmission of nociceptive information from the dorsal horn of the spinal cord to brain areas after the intraplantar injection of CFA. Moreover, dynorphin A within the spinal cord is directly involved in the early phase of an inflammatory pain-like state through NMDA receptors. This study is the first to clarify the ongoing brain activation in the early phase of inflammatory pain associated with an endogenous dynorphinergic pathway in an animal model of pain by means of pharmacological neuroimaging technology.
The authors thank Kazuhiko Miyashita, B.S., and Atsuo Suzuki, B.S. (Graduate Students, Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Shinagawa-ku, Tokyo, Japan), for their expert technical assistance.
References
Chavkin C, James IF, Goldstein A: Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science 1982; 215:413–5Chavkin, C James, IF Goldstein, A
Civelli O, Douglass J, Goldstein A, Herbert E: Sequence and expression of the rat prodynorphin gene. Proc Natl Acad Sci U S A 1985; 82:4291–5Civelli, O Douglass, J Goldstein, A Herbert, E
Laughlin TM, Larson AA, Wilcox GL: Mechanisms of induction of persistent nociception by dynorphin. J Pharmacol Exp Ther 2001; 299:6–11Laughlin, TM Larson, AA Wilcox, GL
Tan-No K, Terenius L, Silberring J, Nylander I: Levels of dynorphin peptides in the central nervous system and pituitary gland of the spontaneously hypertensive rat. Neurochem Int 1997; 31:27–32Tan-No, K Terenius, L Silberring, J Nylander, I
Watson SJ, Khachaturian H, Akil H, Coy DH, Goldstein A: Comparison of the distribution of dynorphin systems and enkephalin systems in brain. Science 1982; 218:1134–6Watson, SJ Khachaturian, H Akil, H Coy, DH Goldstein, A
Ruda MA, Iadarola MJ, Cohen LV, Young WS III: In situ hybridization histochemistry and immunocytochemistry reveal an increase in spinal dynorphin biosynthesis in a rat model of peripheral inflammation and hyperalgesia. Proc Natl Acad Sci U S A 1988; 85:622–6Ruda, MA Iadarola, MJ Cohen, LV Young, WS
Bian D, Ossipov MH, Ibrahim M, Raffa RB, Tallarida RJ, Malan TP Jr, Lai J, Porreca F: Loss of antiallodynic and antinociceptive spinal/supraspinal morphine synergy in nerve-injured rats: Restoration by MK-801 or dynorphin antiserum. Brain Res 1999; 831:55–63Bian, D Ossipov, MH Ibrahim, M Raffa, RB Tallarida, RJ Malan, TP Lai, J Porreca, F
Dubner R, Ruda MA: Activity-dependent neuronal plasticity following tissue injury and inflammation. Trends Neurosci 1992; 15:96–103Dubner, R Ruda, MA
Iadarola MJ, Douglass J, Civelli O, Naranjo JR: Differential activation of spinal cord dynorphin and enkephalin neurons during hyperalgesia: Evidence using cDNA hybridization. Brain Res 1988; 455:205–12Iadarola, MJ Douglass, J Civelli, O Naranjo, JR
Malan TP, Ossipov MH, Gardell LR, Ibrahim M, Bian D, Lai J, Porreca F: Extraterritorial neuropathic pain correlates with multisegmental elevation of spinal dynorphin in nerve-injured rats. Pain 2000; 86:185–94Malan, TP Ossipov, MH Gardell, LR Ibrahim, M Bian, D Lai, J Porreca, F
Kajander KC, Sahara Y, Iadarola MJ, Bennett GJ: Dynorphin increases in the dorsal spinal cord in rats with a painful peripheral neuropathy. Peptides 1990; 11:719–28Kajander, KC Sahara, Y Iadarola, MJ Bennett, GJ
Wagner R, DeLeo JA, Coombs DW, Willenbring S, Fromm C: Spinal dynorphin immunoreactivity increases bilaterally in a neuropathic pain model. Brain Res 1993; 629:323–6Wagner, R DeLeo, JA Coombs, DW Willenbring, S Fromm, C
Peters CM, Lindsay TH, Pomonis JD, Luger NM, Ghilardi JR, Sevcik MA, Mantyh PW: Endothelin and the tumorigenic component of bone cancer pain. Neuroscience 2004; 126:1043–52Peters, CM Lindsay, TH Pomonis, JD Luger, NM Ghilardi, JR Sevcik, MA Mantyh, PW
Vanderah TW, Gardell LR, Burgess SE, Ibrahim M, Dogrul A, Zhong CM, Zhang ET, Malan TP Jr, Ossipov MH, Lai J, Porreca F: Dynorphin promotes abnormal pain and spinal opioid antinociceptive tolerance. J Neurosci 2000; 20:7074–9Vanderah, TW Gardell, LR Burgess, SE Ibrahim, M Dogrul, A Zhong, CM Zhang, ET Malan, TP Ossipov, MH Lai, J Porreca, F
Hauser KF, Foldes JK, Turbek CS: Dynorphin A (1–13) neurotoxicity in vitro: Opioid and non-opioid mechanisms in mouse spinal cord neurons. Exp Neurol 1999; 160:361–75Hauser, KF Foldes, JK Turbek, CS
Stevens CW, Yaksh TL: Dynorphin A and related peptides administered intrathecally in the rat: A search for putative kappa opiate receptor activity. J Pharmacol Exp Ther 1986; 238:833–8Stevens, CW Yaksh, TL
Tan-No K, Takahashi H, Nakagawasai O, Niijima F, Sato T, Satoh S, Sakurada S, Marinova Z, Yakovleva T, Bakalkin G, Terenius L, Tadano T: Pronociceptive role of dynorphins in uninjured animals: N  -ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation. Pain 2005; 113:301–9Tan-No, K Takahashi, H Nakagawasai, O Niijima, F Sato, T Satoh, S Sakurada, S Marinova, Z Yakovleva, T Bakalkin, G Terenius, L Tadano, T
Laughlin TM, Vanderah TW, Lashbrook J, Nichols ML, Ossipov M, Porreca F, Wilcox GL: Spinally administered dynorphin A produces long-lasting allodynia: Involvement of NMDA but not opioid receptors. Pain 1997; 72:253–60Laughlin, TM Vanderah, TW Lashbrook, J Nichols, ML Ossipov, M Porreca, F Wilcox, GL
Vanderah TW, Laughlin T, Lashbrook JM, Nichols ML, Wilcox GL, Ossipov MH, Malan TP Jr, Porreca F: Single intrathecal injections of dynorphin A or des-Tyr-dynorphins produce long-lasting allodynia in rats: Blockade by MK-801 but not naloxone. Pain 1996; 68:275–81Vanderah, TW Laughlin, T Lashbrook, JM Nichols, ML Wilcox, GL Ossipov, MH Malan, TP Porreca, F
Honore P, Rogers SD, Schwei MJ, Salak-Johnson JL, Luger NM, Sabino MC, Clohisy DR, Mantyh PW: Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons. Neuroscience 2000; 98:585–98Honore, P Rogers, SD Schwei, MJ Salak-Johnson, JL Luger, NM Sabino, MC Clohisy, DR Mantyh, PW
Zhang RX, Lao L, Qiao JT, Malsnee K, Ruda MA: Endogenous and exogenous glucocorticoid suppresses up-regulation of preprodynorphin mRNA and hyperalgesia in rats with peripheral inflammation. Neurosci Lett 2004; 359:85–8Zhang, RX Lao, L Qiao, JT Malsnee, K Ruda, MA
Becerra LR, Breiter HC, Stojanovic M, Fishman S, Edwards A, Comite AR, Gonzalez RG, Borsook D: Human brain activation under controlled thermal stimulation and habituation to noxious heat: An fMRI study. Magn Reson Med 1999; 41:1044–57Becerra, LR Breiter, HC Stojanovic, M Fishman, S Edwards, A Comite, AR Gonzalez, RG Borsook, D
Tracey I, Becerra L, Chang I, Breiter H, Jenkins L, Borsook D, Gonzalez RG: Noxious hot and cold stimulation produce common patterns of brain activation in humans: A functional magnetic resonance imaging study. Neurosci Lett 2000; 288:159–62Tracey, I Becerra, L Chang, I Breiter, H Jenkins, L Borsook, D Gonzalez, RG
Wise RG, Rogers R, Painter D, Bantick S, Ploghaus A, Williams P, Rapeport G, Tracey I: Combining fMRI with a pharmacokinetic model to determine which brain areas activated by painful stimulation are specifically modulated by remifentanil. Neuroimage 2002; 16:999–1014Wise, RG Rogers, R Painter, D Bantick, S Ploghaus, A Williams, P Rapeport, G Tracey, I
Wise RG, Williams P, Tracey I: Using fMRI to quantify the time dependence of remifentanil analgesia in the human brain. Neuropsychopharmacology 2004; 29:626–35Wise, RG Williams, P Tracey, I
Leslie RA, James MF: Pharmacological magnetic resonance imaging: A new application for functional MRI. Trends Pharmacol Sci 2000; 21:314–8Leslie, RA James, MF
Honey GD, Corlett PR, Absalom AR, Lee M, Pomarol-Clotet E, Murray GK, McKenna PJ, Bullmore ET, Menon DK, Fletcher PC: Individual differences in psychotic effects of ketamine are predicted by brain function measured under placebo. J Neurosci 2008; 28:6295–303Honey, GD Corlett, PR Absalom, AR Lee, M Pomarol-Clotet, E Murray, GK McKenna, PJ Bullmore, ET Menon, DK Fletcher, PC
Malisza KL, Docherty JC: Capsaicin as a source for painful stimulation in functional MRI. J Magn Reson Imaging 2001; 14:341–7Malisza, KL Docherty, JC
Shih YY, Chen YY, Chen CC, Chen JC, Chang C, Jaw FS: Whole-brain functional magnetic resonance imaging mapping of acute nociceptive responses induced by formalin in rats using atlas registration-based event-related analysis. J Neurosci Res 2008; 86:1801–11Shih, YY Chen, YY Chen, CC Chen, JC Chang, C Jaw, FS
Niikura K, Kobayashi Y, Okutsu D, Furuya M, Kawano K, Maitani Y, Suzuki T, Narita M: Implication of spinal protein kinase Cgamma isoform in activation of the mouse brain by intrathecal injection of the protein kinase C activator phorbol 12,13-dibutyrate using functional magnetic resonance imaging analysis. Neurosci Lett 2008; 433:6–10Niikura, K Kobayashi, Y Okutsu, D Furuya, M Kawano, K Maitani, Y Suzuki, T Narita, M
Hylden JL, Wilcox GL: Intrathecal morphine in mice: A new technique. Eur J Pharmacol 1980; 67:313–6Hylden, JL Wilcox, GL
Ohsawa M, Narita M, Mizoguchi H, Suzuki T, Tseng LF: Involvement of spinal protein kinase C in thermal hyperalgesia evoked by partial sciatic nerve ligation, but not by inflammation in the mouse. Eur J Pharmacol 2000; 403:81–5Ohsawa, M Narita, M Mizoguchi, H Suzuki, T Tseng, LF
Narita M, Shimamura M, Imai S, Kubota C, Yajima Y, Takagi T, Shiokawa M, Inoue T, Suzuki M, Suzuki T: Role of interleukin-1beta and tumor necrosis factor-alpha-dependent expression of cyclooxygenase-2 mRNA in thermal hyperalgesia induced by chronic inflammation in mice. Neuroscience 2008; 152:477–86Narita, M Shimamura, M Imai, S Kubota, C Yajima, Y Takagi, T Shiokawa, M Inoue, T Suzuki, M Suzuki, T
Breese NM, George AC, Pauers LE, Stucky CL: Peripheral inflammation selectively increases TRPV1 function in IB4-positive sensory neurons from adult mouse. Pain 2005; 115:37–49Breese, NM George, AC Pauers, LE Stucky, CL
Tuor UI, Malisza K, Foniok T, Papadimitropoulos R, Jarmasz M, Somorjai R, Kozlowski P: Functional magnetic resonance imaging in rats subjected to intense electrical and noxious chemical stimulation of the forepaw. Pain 2000; 87:315–24Tuor, UI Malisza, K Foniok, T Papadimitropoulos, R Jarmasz, M Somorjai, R Kozlowski, P
Franklin KBJ, Paxions G: The Mouse Brain in Stereotaxic Coordinates. California, Academic Press, 1997Franklin, KBJ Paxions, G California Academic Press
Narita M, Mizoguchi H, Suzuki T, Dun NJ, Imai S, Yajima Y, Nagase H, Tseng LF: Enhanced mu-opioid responses in the spinal cord of mice lacking protein kinase Cgamma isoform. J Biol Chem 2001; 276:15409–14Narita, M Mizoguchi, H Suzuki, T Dun, NJ Imai, S Yajima, Y Nagase, H Tseng, LF
Narita M, Hashimoto K, Amano T, Niikura K, Nakamura A, Suzuki T: Post-synaptic action of morphine on glutamatergic neuronal transmission related to the descending antinociceptive pathway in the rat thalamus. J Neurochem 2008; 104:469–78Narita, M Hashimoto, K Amano, T Niikura, K Nakamura, A Suzuki, T
Wang Z, Gardell LR, Ossipov MH, Vanderah TW, Brennan MB, Hochgeschwender U, Hruby VJ, Malan TP Jr, Lai J, Porreca F: Pronociceptive actions of dynorphin maintain chronic neuropathic pain. J Neurosci 2001; 21:1779–86Wang, Z Gardell, LR Ossipov, MH Vanderah, TW Brennan, MB Hochgeschwender, U Hruby, VJ Malan, TP Lai, J Porreca, F
Jones AK, Brown WD, Friston KJ, Qi LY, Frackowiak RS: Cortical and subcortical localization of response to pain in man using positron emission tomography. Proc Biol Sci 1991; 244:39–44Jones, AK Brown, WD Friston, KJ Qi, LY Frackowiak, RS
Talbot JD, Marrett S, Evans AC, Meyer E, Bushnell MC, Duncan GH: Multiple representations of pain in human cerebral cortex. Science 1991; 251:1355–8Talbot, JD Marrett, S Evans, AC Meyer, E Bushnell, MC Duncan, GH
Borsook D, Moulton EA, Schmidt KF, Becerra LR: Neuroimaging revolutionizes therapeutic approaches to chronic pain. Mol Pain 2007; 3:25Borsook, D Moulton, EA Schmidt, KF Becerra, LR
Treede RD, Kenshalo DR, Gracely RH, Jones AK: The cortical representation of pain. Pain 1999; 79:105–11Treede, RD Kenshalo, DR Gracely, RH Jones, AK
Melzack R: From the gate to the neuromatrix. Pain 1999; Suppl 6:S121–6Melzack, R
Rorden C, Karnath HO: Using human brain lesions to infer function: a relic from a past era in the fMRI age? Nat Rev Neurosci 2004; 5:813–9Rorden, C Karnath, HO
Riley RC, Zhao ZQ, Duggan AW: Spinal release of immunoreactive dynorphin A(1–8) with the development of peripheral inflammation in the rat. Brain Res 1996; 710:131–42Riley, RC Zhao, ZQ Duggan, AW
Iadarola MJ, Brady LS, Draisci G, Dubner R: Enhancement of dynorphin gene expression in spinal cord following experimental inflammation: stimulus specificity, behavioral parameters and opioid receptor binding. Pain 1988; 35:313–26Iadarola, MJ Brady, LS Draisci, G Dubner, R
Zhang RX, Lao L, Qiao JT, Ruda MA: Strain differences in pain sensitivity and expression of preprodynorphin mRNA in rats following peripheral inflammation. Neurosci Lett 2003; 353:213–6Zhang, RX Lao, L Qiao, JT Ruda, MA
Zhu X, Vincler MA, Parker R, Eisenach JC: Spinal cord dynorphin expression increases, but does not drive microglial prostaglandin production or mechanical hypersensitivity after incisional surgery in rats. Pain 2006; 125:43–52Zhu, X Vincler, MA Parker, R Eisenach, JC
Carrion AM, Mellstrom B, Luckman SM, Naranjo JR: Stimulus-specific hierarchy of enhancer elements within the rat prodynorphin promoter. J Neurochem 1998; 70:914–21Carrion, AM Mellstrom, B Luckman, SM Naranjo, JR
Costigan M, Woolf CJ: No DREAM, no pain closing the spinal gate cell 2002; 108:297–300Costigan, M Woolf, CJ
Cole RL, Konradi C, Douglass J, Hyman SE: Neuronal adaptation to amphetamine and dopamine: molecular mechanisms of prodynorphin gene regulation in rat striatum. Neuron 1995; 14:813–23Cole, RL Konradi, C Douglass, J Hyman, SE
Naranjo JR, Mellstrom B, Achaval M, Sassone-Corsi P: Molecular pathways of pain: Fos/Jun-mediated activation of a noncanonical AP-1 site in the prodynorphin gene. Neuron 1991; 6:607–17Naranjo, JR Mellstrom, B Achaval, M Sassone-Corsi, P
Cheng HY, Pitcher GM, Laviolette SR, Whishaw IQ, Tong KI, Kockeritz LK, Wada T, Joza NA, Crackower M, Goncalves J, Sarosi I, Woodgett JR, Oliveira-dos-Santos AJ, Ikura M, van der Kooy D, Salter MW, Penninger JM: DREAM is a critical transcriptional repressor for pain modulation. Cell 2002; 108:31–43Cheng, HY Pitcher, GM Laviolette, SR Whishaw, IQ Tong, KI Kockeritz, LK Wada, T Joza, NA Crackower, M Goncalves, J Sarosi, I Woodgett, JR Oliveira-dos-Santos, AJ Ikura, M van der Kooy, D Salter, MW Penninger, JM
Zhang RX, Ruda MA, Qiao JT: Pre-emptive intrathecal Mk-801, a non-competitive N  -methyl-d-aspartate receptor antagonist, inhibits the up-regulation of spinal dynorphin mRNA and hyperalgesia in a rat model of chronic inflammation. Neurosci Lett 1998; 241:57–60Zhang, RX Ruda, MA Qiao, JT
Gsell W, Burke M, Wiedermann D, Bonvento G, Silva AC, Dauphin F, Buhrle C, Hoehn M, Schwindt W: Differential effects of NMDA and AMPA glutamate receptors on functional magnetic resonance imaging signals and evoked neuronal activity during forepaw stimulation of the rat. J Neurosci 2006; 26:8409–16Gsell, W Burke, M Wiedermann, D Bonvento, G Silva, AC Dauphin, F Buhrle, C Hoehn, M Schwindt, W
Fig. 1. Effect of an intraplantar injection of complete Freund's adjuvant (CFA) on spontaneous pain-like behaviors in wild-type (+/+) and prodynorphin (PDYN) knockout (−/−) mice. Immediately after intraplantar injection, the number of flinching (A-i  ) or licking (A-ii  ) behaviors was counted, and the total duration of flinching and licking behaviors was measured (B  ) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P  < 0.001, wild-type mouse with intraplantar injection of saline versus  wild-type mouse with intraplantar injection of CFA; $$ P  < 0.01, $$$ P  < 0.001, wild-type mouse with intraplantar injection of CFA versus  PDYN knockout mouse with intraplantar injection of CFA.
Fig. 1. Effect of an intraplantar injection of complete Freund's adjuvant (CFA) on spontaneous pain-like behaviors in wild-type (+/+) and prodynorphin (PDYN) knockout (−/−) mice. Immediately after intraplantar injection, the number of flinching (A-i 
	) or licking (A-ii 
	) behaviors was counted, and the total duration of flinching and licking behaviors was measured (B 
	) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P 
	< 0.001, wild-type mouse with intraplantar injection of saline versus 
	wild-type mouse with intraplantar injection of CFA; $$ P 
	< 0.01, $$$ P 
	< 0.001, wild-type mouse with intraplantar injection of CFA versus 
	PDYN knockout mouse with intraplantar injection of CFA.
Fig. 1. Effect of an intraplantar injection of complete Freund's adjuvant (CFA) on spontaneous pain-like behaviors in wild-type (+/+) and prodynorphin (PDYN) knockout (−/−) mice. Immediately after intraplantar injection, the number of flinching (A-i  ) or licking (A-ii  ) behaviors was counted, and the total duration of flinching and licking behaviors was measured (B  ) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P  < 0.001, wild-type mouse with intraplantar injection of saline versus  wild-type mouse with intraplantar injection of CFA; $$ P  < 0.01, $$$ P  < 0.001, wild-type mouse with intraplantar injection of CFA versus  PDYN knockout mouse with intraplantar injection of CFA.
×
Fig. 2. Time course of the effect of intraplantar injection of complete Freund's adjuvant (CFA) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial halamic region (mTH), and lateral thalamic region (lTH) in wild-type (WT) and prodynorphin (PDYN) knockout (KO) mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 6 h (A-i  : CG, B-i  : S1, C-i  : IC, D-i  ; mTH, E-i  ; lTH) after intraplantar injection of CFA in WT and KO mice. Intraplantar injection of CFA produced a significant increase in BOLD signal in the CG (A-ii  ), S1 (B-ii  ), IC (C-ii  ), mTH (D-ii  ), and lTH (E-ii  ) of wild-type mice. Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice.
Fig. 2. Time course of the effect of intraplantar injection of complete Freund's adjuvant (CFA) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial halamic region (mTH), and lateral thalamic region (lTH) in wild-type (WT) and prodynorphin (PDYN) knockout (KO) mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 6 h (A-i 
	: CG, B-i 
	: S1, C-i 
	: IC, D-i 
	; mTH, E-i 
	; lTH) after intraplantar injection of CFA in WT and KO mice. Intraplantar injection of CFA produced a significant increase in BOLD signal in the CG (A-ii 
	), S1 (B-ii 
	), IC (C-ii 
	), mTH (D-ii 
	), and lTH (E-ii 
	) of wild-type mice. Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice.
Fig. 2. Time course of the effect of intraplantar injection of complete Freund's adjuvant (CFA) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial halamic region (mTH), and lateral thalamic region (lTH) in wild-type (WT) and prodynorphin (PDYN) knockout (KO) mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 6 h (A-i  : CG, B-i  : S1, C-i  : IC, D-i  ; mTH, E-i  ; lTH) after intraplantar injection of CFA in WT and KO mice. Intraplantar injection of CFA produced a significant increase in BOLD signal in the CG (A-ii  ), S1 (B-ii  ), IC (C-ii  ), mTH (D-ii  ), and lTH (E-ii  ) of wild-type mice. Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice.
×
Fig. 3. Effect of intrathecal pretreatment with antiserum against dynorphin A (1–17) on pain-like behaviors evoked by intraplantar injection of complete Freund's adjuvant (CFA). Groups of mice were treated intrathecal with antiserum against dynorphin A (1–17) (Dynorphin A/S; 1:100) or control serum 30 min before the intraplantar injection of CFA. Immediately after the intraplantar injection of saline or CFA, the number of flinching (A-i  ) or licking (A-ii  ) behaviors was counted, and the duration of flinching and licking behaviors was measured (B  ) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P  < 0.001, control serum-saline group versus  control serum-CFA group; $$$ P  < 0.001, control serum-CFA group versus  Dynorphin A/S-CFA group.
Fig. 3. Effect of intrathecal pretreatment with antiserum against dynorphin A (1–17) on pain-like behaviors evoked by intraplantar injection of complete Freund's adjuvant (CFA). Groups of mice were treated intrathecal with antiserum against dynorphin A (1–17) (Dynorphin A/S; 1:100) or control serum 30 min before the intraplantar injection of CFA. Immediately after the intraplantar injection of saline or CFA, the number of flinching (A-i 
	) or licking (A-ii 
	) behaviors was counted, and the duration of flinching and licking behaviors was measured (B 
	) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P 
	< 0.001, control serum-saline group versus 
	control serum-CFA group; $$$ P 
	< 0.001, control serum-CFA group versus 
	Dynorphin A/S-CFA group.
Fig. 3. Effect of intrathecal pretreatment with antiserum against dynorphin A (1–17) on pain-like behaviors evoked by intraplantar injection of complete Freund's adjuvant (CFA). Groups of mice were treated intrathecal with antiserum against dynorphin A (1–17) (Dynorphin A/S; 1:100) or control serum 30 min before the intraplantar injection of CFA. Immediately after the intraplantar injection of saline or CFA, the number of flinching (A-i  ) or licking (A-ii  ) behaviors was counted, and the duration of flinching and licking behaviors was measured (B  ) for 20 min after the injection of CFA into the right hind paw of mice. Each point indicates the mean ± SEM of 3–5 mice. Bonferroni test: ***P  < 0.001, control serum-saline group versus  control serum-CFA group; $$$ P  < 0.001, control serum-CFA group versus  Dynorphin A/S-CFA group.
×
Fig. 4. (A  ) Representative reverse transcription–polymerase chain reaction for prodynorphin (PDYN) messenger RNAs on the ipsilateral side of spinal cords obtained from saline-or complete Freund's adjuvant (CFA)-injected mice. Spinal cord samples were prepared at 1, 3, and 6 h after saline or CFA injection (B  ). The intensity of the bands was determined semiquantitatively using ImageJ (National Institutes of Health). The values for PDYN messenger RNA were normalized by the value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA. The value in CFA-injected mice is expressed as a percentage of the increase in saline-injected mice. Each column represents the mean ± SEM of three samples. Student t  test: **P  < 0.01, ***P  < 0.001, versus  saline group. (C  ) Quantitative analysis of PDYN messenger RNA on the spinal cord of saline-or CFA-treated mice at 6 h after the injection. (C-i  ) Amplification plots of fluorescence intensities versus  PCR cycle numbers in each sample are displayed. (C-ii  ) Each column represents the mean ± SEM of three samples. *P  < 0.05 versus  saline-treated group. (D  ) Change in dynorphin A (1–17)-like immunoreactivity on the superficial laminae of the ipsilateral dorsal horn after CFA injection. Photomicrographs show immunofluorescent staining of dynorphin A (1–17) on the superficial layers of the ipsilateral spinal dorsal horn at 6 h after saline (D-i  ) or CFA injection. Dynorphin A-like immunoreactivity observed on the superficial laminae of the ipsilateral dorsal horn after CFA injection (D-ii  ) was significantly increased compared with that after saline injection (D-i  ). Scale bars = 50 μ m.
Fig. 4. (A 
	) Representative reverse transcription–polymerase chain reaction for prodynorphin (PDYN) messenger RNAs on the ipsilateral side of spinal cords obtained from saline-or complete Freund's adjuvant (CFA)-injected mice. Spinal cord samples were prepared at 1, 3, and 6 h after saline or CFA injection (B 
	). The intensity of the bands was determined semiquantitatively using ImageJ (National Institutes of Health). The values for PDYN messenger RNA were normalized by the value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA. The value in CFA-injected mice is expressed as a percentage of the increase in saline-injected mice. Each column represents the mean ± SEM of three samples. Student t 
	test: **P 
	< 0.01, ***P 
	< 0.001, versus 
	saline group. (C 
	) Quantitative analysis of PDYN messenger RNA on the spinal cord of saline-or CFA-treated mice at 6 h after the injection. (C-i 
	) Amplification plots of fluorescence intensities versus 
	PCR cycle numbers in each sample are displayed. (C-ii 
	) Each column represents the mean ± SEM of three samples. *P 
	< 0.05 versus 
	saline-treated group. (D 
	) Change in dynorphin A (1–17)-like immunoreactivity on the superficial laminae of the ipsilateral dorsal horn after CFA injection. Photomicrographs show immunofluorescent staining of dynorphin A (1–17) on the superficial layers of the ipsilateral spinal dorsal horn at 6 h after saline (D-i 
	) or CFA injection. Dynorphin A-like immunoreactivity observed on the superficial laminae of the ipsilateral dorsal horn after CFA injection (D-ii 
	) was significantly increased compared with that after saline injection (D-i 
	). Scale bars = 50 μ m.
Fig. 4. (A  ) Representative reverse transcription–polymerase chain reaction for prodynorphin (PDYN) messenger RNAs on the ipsilateral side of spinal cords obtained from saline-or complete Freund's adjuvant (CFA)-injected mice. Spinal cord samples were prepared at 1, 3, and 6 h after saline or CFA injection (B  ). The intensity of the bands was determined semiquantitatively using ImageJ (National Institutes of Health). The values for PDYN messenger RNA were normalized by the value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA. The value in CFA-injected mice is expressed as a percentage of the increase in saline-injected mice. Each column represents the mean ± SEM of three samples. Student t  test: **P  < 0.01, ***P  < 0.001, versus  saline group. (C  ) Quantitative analysis of PDYN messenger RNA on the spinal cord of saline-or CFA-treated mice at 6 h after the injection. (C-i  ) Amplification plots of fluorescence intensities versus  PCR cycle numbers in each sample are displayed. (C-ii  ) Each column represents the mean ± SEM of three samples. *P  < 0.05 versus  saline-treated group. (D  ) Change in dynorphin A (1–17)-like immunoreactivity on the superficial laminae of the ipsilateral dorsal horn after CFA injection. Photomicrographs show immunofluorescent staining of dynorphin A (1–17) on the superficial layers of the ipsilateral spinal dorsal horn at 6 h after saline (D-i  ) or CFA injection. Dynorphin A-like immunoreactivity observed on the superficial laminae of the ipsilateral dorsal horn after CFA injection (D-ii  ) was significantly increased compared with that after saline injection (D-i  ). Scale bars = 50 μ m.
×
Fig. 5. Effect of a single intrathecal injection of dynorphin A (1–17) on pain-like behavior. Groups of mice were treated intrathecal injection with dynorphin A (1–17) (0.3, 3, 30 pmol) or saline. Immediately after intrathecal injection, the number of licking or biting behaviors was counted (A  ), and the duration of licking or biting behaviors was measured (B  ) for 20 min after the intrathecal injection of dynorphin A (1–17). The intrathecal injection of dynorphin A (1–17) produced a dose-dependent increase in pain-like behaviors compared with control mice injected intrathecal with vehicle. Each point indicates the mean ± SEM of four to six mice. Dunnett test: *P  < 0.05, **P  < 0.01, ***P  < 0.001, versus  intrathecal vehicle group.
Fig. 5. Effect of a single intrathecal injection of dynorphin A (1–17) on pain-like behavior. Groups of mice were treated intrathecal injection with dynorphin A (1–17) (0.3, 3, 30 pmol) or saline. Immediately after intrathecal injection, the number of licking or biting behaviors was counted (A 
	), and the duration of licking or biting behaviors was measured (B 
	) for 20 min after the intrathecal injection of dynorphin A (1–17). The intrathecal injection of dynorphin A (1–17) produced a dose-dependent increase in pain-like behaviors compared with control mice injected intrathecal with vehicle. Each point indicates the mean ± SEM of four to six mice. Dunnett test: *P 
	< 0.05, **P 
	< 0.01, ***P 
	< 0.001, versus 
	intrathecal vehicle group.
Fig. 5. Effect of a single intrathecal injection of dynorphin A (1–17) on pain-like behavior. Groups of mice were treated intrathecal injection with dynorphin A (1–17) (0.3, 3, 30 pmol) or saline. Immediately after intrathecal injection, the number of licking or biting behaviors was counted (A  ), and the duration of licking or biting behaviors was measured (B  ) for 20 min after the intrathecal injection of dynorphin A (1–17). The intrathecal injection of dynorphin A (1–17) produced a dose-dependent increase in pain-like behaviors compared with control mice injected intrathecal with vehicle. Each point indicates the mean ± SEM of four to six mice. Dunnett test: *P  < 0.05, **P  < 0.01, ***P  < 0.001, versus  intrathecal vehicle group.
×
Fig. 6. Time course of the effects of intrathecal injection of dynorphin A (1–17) (30 pmol) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial thalamic region (mTH), and lateral thalamic region (lTH) in mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 2 h (A-i  : CG), 0 or 1 h (B-i  : S1, C-i  : IC, D-i  ; mTH), and 0 or 0.5 h (E-i  : lTH) after the intrathecal injection of dynorphin A (1–17) in mice. The intrathecal injection of dynorphin A (1–17) produced a significant increase in BOLD signal compared with basal intensity (A-ii  : CG; B-ii  : S1; C-ii  : IC; D-ii  mTH; E-ii  : lTH). Each point indicates the mean ± SEM for five mice. CFA = complete Freund's adjuvant.
Fig. 6. Time course of the effects of intrathecal injection of dynorphin A (1–17) (30 pmol) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial thalamic region (mTH), and lateral thalamic region (lTH) in mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 2 h (A-i 
	: CG), 0 or 1 h (B-i 
	: S1, C-i 
	: IC, D-i 
	; mTH), and 0 or 0.5 h (E-i 
	: lTH) after the intrathecal injection of dynorphin A (1–17) in mice. The intrathecal injection of dynorphin A (1–17) produced a significant increase in BOLD signal compared with basal intensity (A-ii 
	: CG; B-ii 
	: S1; C-ii 
	: IC; D-ii 
	mTH; E-ii 
	: lTH). Each point indicates the mean ± SEM for five mice. CFA = complete Freund's adjuvant.
Fig. 6. Time course of the effects of intrathecal injection of dynorphin A (1–17) (30 pmol) on blood oxygenation level-dependent (BOLD) signal intensity in the cingulate cortex (CG), somatosensory cortex (S1), insula cortex (IC), medial thalamic region (mTH), and lateral thalamic region (lTH) in mice. Representative activation maps (overlaid on anatomy) correspond to composite images at 0 or 2 h (A-i  : CG), 0 or 1 h (B-i  : S1, C-i  : IC, D-i  ; mTH), and 0 or 0.5 h (E-i  : lTH) after the intrathecal injection of dynorphin A (1–17) in mice. The intrathecal injection of dynorphin A (1–17) produced a significant increase in BOLD signal compared with basal intensity (A-ii  : CG; B-ii  : S1; C-ii  : IC; D-ii  mTH; E-ii  : lTH). Each point indicates the mean ± SEM for five mice. CFA = complete Freund's adjuvant.
×
Fig. 7. Effect of intrathecal pretreatment with MK-801 on dynorphin A (1–17)-mediated pain-like response. Groups of mice were treated intrathecal with a noncompetitive N  -methyl-d-a spartic acid receptor antagonist, MK-801 (1 nmol/mouse), or vehicle 30 min before the intrathecal injection of dynorphin A (1–17) (30 pmol). Immediately after the intrathecal injection of dynorphin A (1–17), the number of licking or biting behaviors was counted (A  ), and the duration of licking or biting behaviors was measured (B  ) for 20 min after the intrathecal injection of dynorphin A (1–17). Dunnett test: $$ P  < 0.01, $$$ P  < 0.001, versus  saline-dynorphin A (1–17) group.
Fig. 7. Effect of intrathecal pretreatment with MK-801 on dynorphin A (1–17)-mediated pain-like response. Groups of mice were treated intrathecal with a noncompetitive N 
	-methyl-d-a spartic acid receptor antagonist, MK-801 (1 nmol/mouse), or vehicle 30 min before the intrathecal injection of dynorphin A (1–17) (30 pmol). Immediately after the intrathecal injection of dynorphin A (1–17), the number of licking or biting behaviors was counted (A 
	), and the duration of licking or biting behaviors was measured (B 
	) for 20 min after the intrathecal injection of dynorphin A (1–17). Dunnett test: $$ P 
	< 0.01, $$$ P 
	< 0.001, versus 
	saline-dynorphin A (1–17) group.
Fig. 7. Effect of intrathecal pretreatment with MK-801 on dynorphin A (1–17)-mediated pain-like response. Groups of mice were treated intrathecal with a noncompetitive N  -methyl-d-a spartic acid receptor antagonist, MK-801 (1 nmol/mouse), or vehicle 30 min before the intrathecal injection of dynorphin A (1–17) (30 pmol). Immediately after the intrathecal injection of dynorphin A (1–17), the number of licking or biting behaviors was counted (A  ), and the duration of licking or biting behaviors was measured (B  ) for 20 min after the intrathecal injection of dynorphin A (1–17). Dunnett test: $$ P  < 0.01, $$$ P  < 0.001, versus  saline-dynorphin A (1–17) group.
×
Fig. 8. The effect of the pretreatment of the MK-801 on increase in dynorphin-induced signal intensity (A  : cingulate cortex (CG); B  : somatosensory cortex (S1); C  : insula cortex (IC); D  : medial thalamic region (mTH); E  : lateral thalamic region (lTH)). Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice. Bonferroni test: **P  < 0.01, ***P  < 0.001, the basal intensity of saline-pretreated groups versus  dynorphin challenge in saline-pretreated groups; $ P  < 0.05, $$ P  < 0.01, $$$ P  < 0.001, the dynorphin challenge in saline-pretreated groups versus  dynorphin challenge in MK-801-pretreated groups.
Fig. 8. The effect of the pretreatment of the MK-801 on increase in dynorphin-induced signal intensity (A 
	: cingulate cortex (CG); B 
	: somatosensory cortex (S1); C 
	: insula cortex (IC); D 
	: medial thalamic region (mTH); E 
	: lateral thalamic region (lTH)). Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice. Bonferroni test: **P 
	< 0.01, ***P 
	< 0.001, the basal intensity of saline-pretreated groups versus 
	dynorphin challenge in saline-pretreated groups; $ P 
	< 0.05, $$ P 
	< 0.01, $$$ P 
	< 0.001, the dynorphin challenge in saline-pretreated groups versus 
	dynorphin challenge in MK-801-pretreated groups.
Fig. 8. The effect of the pretreatment of the MK-801 on increase in dynorphin-induced signal intensity (A  : cingulate cortex (CG); B  : somatosensory cortex (S1); C  : insula cortex (IC); D  : medial thalamic region (mTH); E  : lateral thalamic region (lTH)). Data are expressed as percentages of the corresponding baseline levels with mean ± SEM for five mice. Bonferroni test: **P  < 0.01, ***P  < 0.001, the basal intensity of saline-pretreated groups versus  dynorphin challenge in saline-pretreated groups; $ P  < 0.05, $$ P  < 0.01, $$$ P  < 0.001, the dynorphin challenge in saline-pretreated groups versus  dynorphin challenge in MK-801-pretreated groups.
×