Characterization of nociceptive response to chemical, mechanical, and thermal stimuli in adolescent rats with neonatal dopamine depletion

Neuroscience 289 (2015) 43–55

CHARACTERIZATION OF NOCICEPTIVE RESPONSE TO CHEMICAL, MECHANICAL, AND THERMAL STIMULI IN ADOLESCENT RATS WITH NEONATAL DOPAMINE DEPLETION M. OGATA, * K. NODA, H. AKITA AND H. ISHIBASHI Department of Physiology, School of Allied Health Sciences, Kitasato University, 1-15-1, Kitasato Minami-ku, Sagamihara, Kanagawa 252-0373, Japan

Key words: nociceptive response, neonatal dopamine depletion, 6-hydroxydopamine, formalin test, dopaminergic neural system, c-Fos.

Abstract—Rats with dopamine depletion caused by 6-hydroxydopamine (6-OHDA) treatment during adulthood and the neonatal period exhibit akinetic motor activity and spontaneous motor hyperactivity during adolescence, respectively, indicating that the behavioral effects of dopamine depletion depend on the period of lesion development. Dopamine depletion during adulthood induces hyperalgesic response to mechanical, thermal, and/or chemical stimuli, whereas the effects of neonatal dopamine depletion on nociceptive response in adolescent rats are yet to be examined. The latter aspect was addressed in this study, and behavioral responses were examined using von-Frey, tail flick, and formalin tests. The formalin test revealed that rats with neonatal dopamine depletion exhibited a significant increase in nociceptive response during interphase (6–15 min post formalin injection) and phase 2 (16–75 min post formalin injection). This increase in nociceptive response to the formalin injection was not reversed by pretreatment with methamphetamine, which ameliorates motor hyperactivity observed in adolescent rats with neonatal 6-OHDA treatment. The von-Frey filament and tail flick tests failed to reveal significant differences in withdrawal thresholds between neonatal 6-OHDA-treated and vehicle-treated rats. The spinal neuronal response to the formalin injection into the rat hind paw was also examined through immunohistochemical analysis of c-Fos protein. Significantly increased numbers of c-Fos-immunoreactive cells were observed in laminae I–II and V–VI of the ipsilateral spinal cord to the site of the formalin injection in rats with neonatal dopamine depletion compared with vehicle-treated rats. These results suggest that the dopaminergic neural system plays a crucial role in the development of a neural network for tonic pain, including the spinal neural circuit for nociceptive transmission, and that the mechanism underlying hyperalgesia to tonic pain is not always consistent with that of spontaneous motor hyperactivity induced by neonatal dopamine depletion. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

INTRODUCTION Previous studies have shown that the dopamine system is not only involved in motor control but also in the modulation and processing of somatosensory information, including pain (Chudler and Dong, 1995; Magnusson and Fisher, 2000). Neurons in the caudateputamen, globus pallidus, and substantia nigra (SN) respond to non-noxious as well as noxious somatosensory stimuli (Romo and Schultz, 1989; Tsai, 1989; Chudler et al., 1993). A previous report showed that dopaminergic neurons in the midbrain receive short-latency nociceptive inputs from the skin via the mesopontine parabrachial nucleus (Coizet et al., 2010). At least 60% of patients with Parkinson’s disease, which is caused by the degeneration of nigrostriatal dopamine pathway, experience certain types of pain such as musculoskeletal, radicular neuropathic, or dystonic pain, in addition to motor abnormalities (Chaudhuri and Schapira, 2009; Broen et al., 2012; Fil et al., 2013). These clinical reports are also supported by experimental data, as detailed below. Rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions in the nigrostriatal dopaminergic neurons show hypersensitivity to mechanical (Saade´ et al., 1997; Takeda et al., 2005) and thermal (Saade´ et al., 1997; Chudler and Lu, 2008) stimuli. Furthermore, unilateral 6-OHDA-treated rats exhibited hyperalgesic response to pain-inducing chemical stimuli (Tassorelli et al., 2007; Chudler and Lu, 2008). The treatment of rodents with 6-OHDA during adulthood or the neonatal period causes dopamine depletion in the central nervous system, but the effects on motor activity depend on the developmental period during which treatment is administered. Treatment during adulthood results in akinetic motor activity, evaluated as a motor symptom in Parkinson’s disease (Schober, 2004; Eskow et al., 2010; Blandini and Armentero, 2012; Blesa et al., 2012), whereas treatment in the neonatal period results in spontaneous motor hyperactivity during adolescence, which has been evaluated as a symptom of attention-deficit hyperactivity disorder (ADHD) (Shaywitz et al., 1976; Zhang et al., 2001;

*Corresponding author. Tel/fax: +81-42-778-8153. E-mail addresses: [email protected] (M. Ogata), [email protected] (K. Noda), [email protected]. ac.jp (H. Akita), [email protected] (H. Ishibashi). Abbreviations: 6-OHDA, 6-hydroxydopamine; ADHD, attention-deficit hyperactivity disorder; ANOVA, analysis of variance; Fos-ir, c-Fos-immunoreactivity; LC, locus coeruleus; MAP, methamphetamine; SN, substantia nigra; TH, tyrosine hydroxylase; VTA, ventral tegmental area. http://dx.doi.org/10.1016/j.neuroscience.2015.01.002 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 43

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Davids et al., 2003; Masuo et al., 2004). Moreover, treatment with L-DOPA ameliorates akinetic motor activity induced by the treatment of adult rats with 6-OHDA (Olsson et al., 1995; Lundblad et al., 2002; Marin et al., 2007) but not the motor hyperactivity induced by the treatment of neonatal rats with 6-OHDA. On the other hand, the administration of psychostimulants such as amphetamine and methamphetamine (MAP), which elicits motor hyperactivity in normal adult rats, produces a sedative effect in the ADHD animal model generated as a consequence of neonatal dopamine depletion (Avale et al., 2004; Masuo et al., 2004). The expression of the c-Fos protein, encoded by the immediate-early c-fos in the spinal dorsal horn, constitutes a marker of neuronal activity induced by several noxious stimuli (Harris, 1998; Buritova et al., 2005). The intraplantar injection of formalin into the hind paw of a rodent evokes the expression of the c-Fos protein chiefly in the superficial (I–II) and deep (V–VI) laminae of the dorsal horn, where the neurons responding to noxious stimuli are localized (Peterson et al., 1997; Jinks et al., 2002; Buritova et al., 2005). Acute noxious stimulation such as that induced by the injection of formalin into the rodent hind paw evokes the expression of c-Fos at 30 min, which peaks at 60–120 min post stimulation (Gao and Ji, 2009). As mentioned above, the effects of dopamine depletion on responses to certain stimuli are dependent on the developmental period during which the lesion was produced. A few reports have also shown that the dopaminergic neuronal system is vulnerable to perinatal stress such as hypoxia and maternal separation (Decker et al., 2003; Jahng et al., 2010). It may be difficult to clinically investigate pain sensitivity in children with ADHD because of ethical reasons. Therefore, although rats with dopamine depletion during adulthood reportedly exhibit hypersensitive responses to certain noxious stimuli, the effects of neonatal dopamine depletion on nociceptive response during adolescence have not been investigated. Recently, hypersensitivity to cold pain and analgesic effects of methylphenidate on pain in adults with ADHD have been reported (Treister et al., 2013). However, it is suggested that data obtained from childhood or adolescence are more relevant for understanding the symptoms of ADHD. In the present study, we investigated the effects of neonatal dopamine depletion on motor activity and responses to noxious somatosensory stimulation during adolescence. For this purpose, we analyzed nociceptive behavioral responses to mechanical, thermal, and chemical stimuli in adolescent rats subjected to neonatal 6-OHDA treatment, and examined whether psychostimulants, known to produce sedative effects pertaining to motor hyperactivity induced by neonatal dopamine depletion reverse the 6-OHDA-induced alteration in nociceptive response in rats with neonatal dopamine depletion. Furthermore, spontaneous locomotor activity of neonatal 6-OHDA-treated rats during adolescence was examined. In addition to the behavioral tests, neuronal activity in the spinal neurons in response to the chemical stimulus was examined in adolescent rats

subjected to neonatal 6-OHDA treatment by monitoring the expression of the c-Fos protein in the spinal cord through histological analysis.

EXPERIMENTAL PROCEDURES Subjects The experimental procedures employed in this study were approved by the Animal Care and Use Committee of Kitasato University, Japan, and are in accordance with the NIH guidelines for the Care and Use of Laboratory Animals. All possible efforts were made to minimize animal suffering and to reduce the number of animals used. Pregnant Wistar Hannover rats (CLEA Japan, Inc., Japan) were obtained at 14 days of gestation and were individually housed in a clear plastic cage containing woodchip bedding material. Rats were housed in a temperature-, humidity-, and light-controlled room with a 12-h light/dark cycle (with the lights turned off at 8:00 p.m.) and allowed ad libitum access to food and water. Animals and drug treatment On postnatal day 4, male rat pups were subcutaneously injected desipramine hydrochloride (25 mg/kg body weight; Sigma, St. Louis, MO, USA) for the protection of the noradrenergic system from neurotoxic effects of 6-OHDA (Sigma). After a time interval of 30 min, the rats subjected to hypothermic anesthesia were placed in a stereotaxic apparatus (Narishige, Tokyo, Japan) with a Neonatal Rat Adaptor and received bilateral intracerebroventricular injections of 6-OHDA (50 lg of free base dissolved in 0.1% ascorbic acid/saline) or the vehicle at the following stereotaxic coordinates: 0.3 mm caudal to the bregma, 1.0 mm lateral to the midline on both sides, and 2.0 mm ventral to the dural surface. Litters from a single dam were divided into two or three groups namely, 6-OHDA-treated, vehicle-treated, and non-treated rats. Following the administration of 6-OHDA or the vehicle, the rats were warmed to normal body temperature with a heat lamp and returned to the dam in the home cage once normal activity was regained. The rats were weaned on the 25th day of postnatal life, and two or three rats subjected to the same treatment were housed per cage. Behavioral experiments Behavioral tests were conducted at 8 weeks of age. Prior to behavioral tests, the rats were weighed and handled daily for 5 min on four consecutive days in order to acclimatize them with the general procedures involved and to promote the stability of behavioral responses. Locomotor activity was assessed using the open field test (Akita et al., 2006). In brief, each rat was placed in the center of the open field (100  100  50 cm, white square box) at the beginning and then allowed to freely explore the field for 30 min. The activity in the open field was recorded with a camera connected to a computerized

M. Ogata et al. / Neuroscience 289 (2015) 43–55

video tracking system (Time OFCR1, O’Hara & Co., Ltd., Japan), and the total locomotion distance was measured as the spontaneous locomotor activity. The von-Frey filament test was conducted to analyze responses to the mechanical stimulus (Noda et al., 2014). In brief, each rat was placed on a wire-mesh floor covered by a plastic box (21  13  15 cm) and adapted to the test environment for at least 15 min. Calibrated von-Frey filaments (2, 3, 5, 8, 10, and 14 g) were applied to the middle-plantar surface of each hind paw. For each filament, five trials with an interval of 30 s were performed per hind paw. The minimum force (g) of the filament that evoked withdrawal of the hind paw in at least three of the five trials was designated as the withdrawal threshold. On the following day (one day post von-Frey filament test), the tail flick test was conducted to analyze responses to the thermal stimulus (Noda et al., 2014). In brief, a beam of radiant heat was focused on the tail of lightly restrained rats, and the latency of the tail withdrawal response to the light beam was automatically recorded (Muromachi Kikai Co., Tokyo, Japan). The trial was terminated at the predetermined cut-off time of 14 s in the absence of a response. The mean value from five trials was calculated as the withdrawal latency. Three days after the tail flick test, the response to the chemical stimulus was assessed using the formalin test (Noda et al., 2014). In brief, a single rat was placed in a Plexiglas observation chamber (33  22  14 cm) and adapted to the test environment for at least 15 min. A large mirror was mounted under the observation chamber to permit the observation of the rat’s hind paws. A 5% solution of formalin (50 ll) was subcutaneously injected into the middle-plantar region of the right hind paw. The rat was then returned to the observation chamber and continuously monitored for 75 min to assess the nociceptive behavior according to a standard scoring method, as detailed below. A nociceptive score was calculated for each 5-min block during the 75-min period on the basis of the time spent in each of the four behavioral categories: 0, full weight placed on the injected paw; 1, reduced weight on the injected paw; 2, elevation of the injected paw; and 3, licking, biting, and shaking of the injected paw. The weighted nociceptive score was calculated as described previously (Zhu et al., 1997), which revealed the nociceptive response to formalin. Furthermore, the effect of administering MAP, which impairs the functions of dopamine and noradrenaline transporters, on nociceptive behavioral response was examined using the formalin test. MAP was dissolved in saline (0.9% w/v NaCl) and administered intraperitoneally, at doses of 1, 2, and 4 mg/kg, 15 min prior to the formalin injection into the right hind paw of rats. The doses of MAP were decided according to reports that showed analgesic or sedative effects of MAP administration on nociceptive responses or motor hyperactivity of neonatal 6-OHDA-treated rats (Masuo et al., 2004; Camarasa et al., 2009; Yamamotova´ et al., 2011). The number of animals used in the experiment was reduced by employing data from the formalin test conducted on neonatal rats treated with 6-OHDA, the vehicle, or left non-treated, as corresponding to 0 mg/kg MAP, and data were then compared with those obtained

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during administration of MAP to rats for deciphering the nociceptive response. Immunohistochemistry and determination of c-Fospositive cell counts Immunostaining was performed as described previously (Ogata et al., 2006). Briefly, after the completion of behavioral experiments, the rats were subjected to deep, prolonged anesthesia through intraperitoneal injection of urethane (1.4 g/kg), followed by transcardial perfusion with 200 ml of heparinized (20 unit/ml) saline and subsequently, with 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Rats were decapitated, and brains were removed, post-fixed overnight in the same fixative, and saturated in 30% sucrose solution at 4 °C for at least 24 h. To evaluate the combined effects of the 6-OHDA injection and pretreatment with desipramine hydrochloride on dopaminergic and noradrenergic neurons, 40-lm-thick coronal sections spanning the striatum, SN, or locus coeruleus (LC) were cut using a sliding microtome and stained for tyrosine hydroxylase (TH). The sections were pre-incubated for l h with 0.1-M phosphate-buffered saline (PBS) containing 4% normal horse serum and 0.3% Triton X-100. Free-floating sections were then incubated overnight with 1:5000 dilution of TH monoclonal antibody (MAB318; Millipore, Temecula, CA, USA) at room temperature. The sections were washed with 0.1 M PBS, and incubated for 1 h at room temperature with a 1:400 dilution of biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA), and then with a 1:100 dilution of the avidin–biotin complex (Elite ABC Kit, Vector Laboratories). The sections were washed with 50 mM PB, incubated with 0.05% diaminobenzidine (Sigma) and 0.004% hydrogen peroxide for 5 min at room temperature, and mounted on subbed slides to visualize immunoreactive cells. The effects of dopamine depletion on the spinal neuronal response evoked by the formalin injection into the hind paw were examined by immunostaining lumbar spinal cord sections with antibody against c-Fos. Two or 4 h following the subcutaneous injection of 5% formalin (50 ll) to the right hind paw, rats subjected to neonatal treatment with the vehicle (2 h, n = 7; 4 h, n = 7) or 6-OHDA (2 h, n = 8; 4 h, n = 9) were anesthetized and perfused, as mentioned above. Frozen transverse sections (40 lm) of the spinal cord were sampled at 120-lm intervals, and immunostained with 1:5000 dilution of the polyclonal c-Fos antibody (sc-52; Santa Cruz Biotechnology, Dallas, TX, USA) and 1:400 dilution of anti-rabbit IgG (Vector Laboratories), as described above. For the quantification of c-Fos-immunoreactive (Fos-ir)-positive cells, sections were first examined at 4 magnification under a light microscope (BX60; Olympus, Tokyo, Japan) for determining the segmental level, and 10 sections containing many Fos-ir-positive cells were selected. Digital images of the selected sections were acquired at 10 magnification with a CCD camera (DP72; Olympus), and the number of Fos-ir-positive cells in the superficial (laminae I–II) and deep (V–VI) laminae of both sides of the spinal cord

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was counted using an image analysis tool (cellSens Dimension; Olympus). Data normalization was performed by selecting four sections with the highest counts and obtaining average Fos-ir-positive neuron counts for each animal. The quantitative analysis of Fos-ir-positive cells was conducted by a blinded investigator to rule out any bias.

Pretreatment with desipramine hydrochloride protected the LC neurons from the neurotoxic effects of the injected 6-OHDA. Thus, no differences in TH immunoreactivity between the three groups were observed in LC (Fig. 1D–F).

Statistical analysis

Locomotor activity in the open field. The open field test was employed to investigate whether the neonatal 6-OHDA-treated rats in the present study showed motor hyperactivity during adolescence. Distances traveled for 30 min in the open field by non-treated, vehicle-treated, and 6-OHDA-treated rats (eight each) were 57.8 ± 5.50 m, 63.0 ± 4.46 m, and 88.3 ± 5.03 m, respectively. A one-way ANOVA revealed significant differences in the distances traveled between the three groups [F(2, 21) = 10.65, p = 0.0006]. Post-hoc analysis with Tukey’s multiple comparison test showed that compared with non-treated and vehicle-treated rats, 6-OHDA-treated rats exhibited a significant increase in the distance traveled (p = 0.0009 vs. non-treated rats, p = 0.0048 vs. vehicle-treated rats).

Data were expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, CA, USA). Data from the open field, the von-Frey filament, and tail flick tests were analyzed using a one-way analysis of variance (ANOVA) to examine differences between non-treated, vehicle-treated, and 6-OHDAtreated rats. In the case of formalin-induced nociceptive behavior, differences between the three groups in the nociceptive scores of each phase were analyzed using a one-way ANOVA, and the time course of changes in nociceptive scores per 5-min intervals and the numbers of Fos-ir-positive cells in the spinal cord 2-h and 4-h time points following the formalin injection were compared using a two-way (drug treatment  time) ANOVA. Post-hoc analysis was performed using either Tukey’s or Bonferroni’s multiple comparison tests, as appropriate. A p-value of <0.05 was considered statistically significant.

RESULTS Tyrosine hydroxylase immunohistochemistry The bilateral cerebroventricular injection of 6-OHDA during the neonatal period resulted in severe loss of TH immunoreactivity on both sides of SN and the ventral tegmental area (VTA; Fig. 1). Dense TH immunoreactivity was observed in SN and VTA of non-treated (Fig. 1A) and vehicle-treated (Fig. 1B) rats, whereas immunoreactivity was absent in SN and was strongly reduced in the VTA of 6-OHDA-treated rats (Fig. 1C).

Behavioral tests

Withdrawal response to mechanical and thermal stimuli. The von-Frey filament test was employed to determine withdrawal thresholds to mechanical stimuli applied on the hind paw on both sides. Significant lateralization of withdrawal thresholds was not detected in any group (data not shown); therefore, data from both hind paws were averaged for determining differences between the groups (Fig. 2A). The withdrawal thresholds of non-treated, vehicle-treated, and 6-OHDA-treated rats (10 each) were 8.78 ± 0.83 g, 10.08 ± 0.66 g, and 8.63 ± 1.02 g, respectively. No significant differences were detected between the three groups in withdrawal thresholds to the mechanical stimulus [Fig. 2A; F(2, 27) = 0.88, p = 0.43]. The tail flick test was employed for assessing withdrawal latency to the thermal stimulus (Fig. 2B). Non-treated, vehicle-treated, and 6-OHDA-treated rats

Fig. 1. Photomicrographs of tyrosine hydroxylase immunoreactive neurons in the midbrain (A-C) and the pons (D-F) of the non-treated rats (A and D), the vehicle-treated rats (B and E) and 6-OHDA-treated rats (C and F). Scale bar = 1000 lm. Abbreviations: SN, substantia nigra; VTA, ventral tegmental area; LC, locus coeruleus.

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Fig. 2. Effects of neonatal dopamine depletion on the withdrawal responses to mechanical stimulation to the hind paw in the von-Frey test (A) and to thermal stimulation to the tail in the tail flick test (B) during adolescence. n = 10 for all groups in both tests.

(10 each) showed withdrawal latencies of 4.10 ± 0.28 s, 4.18 ± 0.26 s, and 4.31 ± 0.47 s, respectively. No significant differences in withdrawal latency to the thermal stimulus were observed between the three groups [Fig. 2B; F(2, 27) = 0.09, p = 0.92]. Nociceptive response to the chemical stimulus. The formalin test was employed to investigate nociceptive responses to the chemical stimulus. The nociceptive behaviors induced by the formalin injection into the right hind paw were assessed as per the nociceptive categories (as described in ‘Behavioral experiments’ of Experimental procedures) and expressed as the nociceptive score for the comparison of responses. The injection of formalin into the right hind paw of nontreated and vehicle-treated rats resulted in the typical biphasic increase in the nociceptive score (Fig. 3A), with an immediate increase following the injection (phase 1, 0–5 min). This immediate increase was followed by a transient period with almost no nociceptive behavior (interphase, 6–15 min) and a long-lasting increase in the nociceptive score lasting approximately 60 min (phase 2, 16–75 min); phase 2 is divisible into periods with sustained (early phase 2, 16–50 min) and decreased (late phase 2, 51–75 min) nociceptive scores. Rats subjected to the 6-OHDA treatment also showed a biphasic time course of the nociceptive score following the formalin injection; however, in these rats, marked increases in the nociceptive score were observed only during the interphase and late phase 2 (Fig. 3A). A twoway repeated measures ANOVA revealed that the treatment, time, and treatment  time interaction significantly affected the nociceptive score induced by the formalin injection [F(2, 27) = 16.10, p < 0.0001; F(14, 378) = 62.20, p < 0.0001; F(28, 378) = 5.93, p < 0.0001, respectively]. Further analysis with Tukey’s multiple comparison test indicated that neonatal treatment with 6-OHDA resulted in significantly increased nociceptive scores at the beginning of interphase (Fig. 3A; 10 min after the formalin injection) and the later time points of late phase 2 (Fig. 3A; 65, 70, and 75 min after the formalin injection) compared with the nontreatment and treatment with the vehicle. The averages of the nociceptive score during phase 1, interphase, early phase 2, and late phase 2 were compared between the three groups (Fig. 3B–E). Significant differences in nociceptive score were observed between the three

groups at interphase, early phase 2, and late phase 2, but not phase 1 [Fig. 3B–E; F(2, 27) = 10.88, p = 0.0003; F(2, 27) = 8.09, p = 0.0018; F(2, 27) = 10.93, p = 0.0003; F(2, 27) = 2.37, p = 0.11, respectively]. Post-hoc analysis with Tukey’s multiple comparison test showed that the 6-OHDA-treated rats exhibited a significant increase in the nociceptive score compared with non-treated and vehicle-treated rats during all the phases of the formalin test, excluding phase 1 (p > 0.01 vs. non-treated rats at interphase, early phase 2, and late phase 2 and vs. vehicle-treated rats at interphase and late phase 2; p > 0.05 vs. vehicle-treated rats at early phase 2). Effect of MAP on nociceptive responses to the chemical stimulus. Hyperalgesic response to the formalin injection into the hind paw was observed in rats with neonatal 6-OHDA treatment; therefore, the effect of pretreatment with MAP, which exhibits sedative effects on the hyperactivity induced by neonatal dopamine depletion, on the nociceptive hyperalgesic response to the formalin injection was assessed. In this experiment, data corresponding to vehicletreated and 6-OHDA-treated rats are presented, because significant differences were not observed in the nociceptive scores between non-treated and vehicletreated rats upon MAP pretreatment (data not shown). Fig. 4 shows the effects of MAP pretreatment on the nociceptive score of vehicle-treated rats in the formalin test. A two-way repeated measures ANOVA revealed that the dose of MAP, the time, and the dose  time interaction significantly affected the time course of the nociceptive score induced by the formalin injection in vehicle-treated rats [F(3, 25) = 27.42, p < 0.0001; F(14, 350) = 66.86, p < 0.0001; F(42, 350) = 1.58, p = 0.015, respectively]. Post-hoc analysis with Tukey’s multiple comparison test showed that compared with the non-MAP-administered rats (n = 10), vehicle-treated rats showed a significant decrease in the nociceptive score at 10 min post formalin injection after the administration of 1 mg/kg MAP (n = 7) and showed a significant decrease in the nociceptive score at 10, 15, 20, 60, and 65 min post formalin injection after the administration of 2 mg/kg MAP (n = 6). Further, the administration of 4 mg/kg MAP (n = 6) significantly decreased the nociceptive score of vehicle-treated rats at all time points during the observation period, compared with rats with 0 mg/kg MAP administration

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Fig. 3. Effects of neonatal dopamine depletion on the behavioral response to chemical stimuli to the hind paw in the formalin test. (A) Time course of nociceptive score in the formalin test. Formalin was injected at time 0 min. (B–E) Effect of neonatal dopamine depletion on total nociceptive score at each phase of formalin test; phase 1, 0–5 min post formalin injection; interphase, 6–15 min post formalin injection; early phase 2, 16–50 min post formalin injection; late phase 2, 51–75 min post formalin injection. n = 10 for all groups. ⁄⁄p < 0.01, compared with non-treated rats. #p < 0.05, ## p < 0.01, compared with vehicle-treated rats.

(Fig. 4A). The inhibitory effects of MAP on the nociceptive score during each phase of the observation period in the formalin test are shown (Fig. 4B–E). Dose-dependent reductions in the nociceptive score induced by the administration of MAP (0, 1.0, 2.0, or 4.0 mg/kg) were observed in all four phases. A one-way ANOVA revealed significant differences in the nociceptive score at each phase [phase 1, F(3, 25) = 44.82, p < 0.0001; interphase, F(3, 25) = 15.45, p < 0.0001; early phase 2, F(3, 25) = 33.50, p < 0.0001; late phase 2, F(3, 25) = 8.03, p = 0.0006] between rats pretreated with various amounts of MAP (0, 1.0, 2.0, and 4.0 mg/kg). Post-hoc analysis with Bonferroni’s multiple comparison test showed a significant reduction in the nociceptive score in phase 1 and interphase, with the administration of 1, 2, and 4 mg/kg of MAP (Fig. 4B, C), and in early

phase 2 and late phase 2, only with high doses of MAP (4 mg/kg; Fig. 4D, E), compared with rats with 0 mg/kg MAP administration. The nociceptive scores of rats administered 4 mg/kg MAP, when expressed as a percentage relative to rats administered 0 mg/kg MAP, were 55.75 ± 3.70%, 18.39 ± 5.66%, 62.25 ± 5.74%, and 34.34 ± 6.72% during phase 1, interphase, early phase 2, and late phase 2, respectively, indicating the strongest effects of MAP pretreatment on nociceptive behavior during the interphase. Fig. 5 shows the effects of intraperitoneal MAP administration on the nociceptive scores of 6-OHDAtreated rats in the formalin test. A two-way repeated measures ANOVA revealed that MAP administration at any dose did not significantly affect the time course of nociceptive score induced by the formalin injection in

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Fig. 4. Effects of MAP pretreatment (0 mg/kg, 1 mg/kg, 2 mg/kg and 4 mg/kg) on the behavioral response to chemical stimuli to the hind paw in the formalin test of vehicle-treated rats. (A) Time course of nociceptive score induced by formalin injection with MAP pretreatment. Formalin was injected at time 0 min. (B–E) Effect of MAP pretreatment on total nociceptive score at each phase of formalin test. Division of phase in the formalin test was the same as in Fig. 3. n = 10, 7, 6 and 6 for 0 mg/kg, 1 mg/kg, 2 mg/kg and 4 mg/kg MAP-treated rats, respectively. ⁄p < 0.05, ⁄⁄p < 0.01, compared with 0 mg/kg MAP-treated rats at each phase.

6-OHDA-treated rats [0 mg/kg, n = 10; 1, 2, 4 mg/kg, n = 6 each; F(3, 24) = 0.54, p = 0.66]. The effect of MAP administration on the nociceptive score during each phase of the formalin test with these rats is shown (Fig. 5B–E). A one-way ANOVA revealed that different doses of MAP failed to exert a significant effect on the nociceptive score at any phase [phase 1, F(3, 24) = 1.24, p = 0.32; interphase, F(3, 24) = 1.04, p = 0.39; early phase 2, F(3, 24) = 2.82, p = 0.06; late phase 2, F(3, 24) = 2.03, p = 0.14].

Expression of c-Fos protein in the spinal cord after formalin injection into rat hind paws Neonatal dopamine depletion resulted in long-lasting nociceptive behavior after the formalin injection into the right hind paw (Fig. 3). To investigate whether abnormal neuronal response to the formalin injection is also observed in the spinal neurons of rats with neonatal dopamine depletion, immunohistochemical analysis was performed, and the number of Fos-ir-positive cells counted in laminae I–II and V–VI of the spinal cord at the

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Fig. 5. Effects of MAP pretreatment (0 mg/kg, 1 mg/kg, 2 mg/kg and 4 mg/kg) on the behavioral response to chemical stimuli to the hind paw in the formalin test of 6-OHDA-treated rats. (A) Time course of nociceptive score induced by formalin injection with MAP pretreatment. Formalin was injected at time 0 min. (B–E) Effect of MAP pretreatment on total nociceptive score at each phase of formalin test. Division of phase in the formalin test was the same as in Fig. 3. n = 10, 6, 6 and 6 for 0 mg/kg, 1 mg/kg, 2 mg/kg and 4 mg/kg MAP-treated rats, respectively.

2-h and 4-h time points following the formalin injection. Fos-ir-positive cells were observed on both sides of the spinal cord, but those on the side contralateral to the site of the formalin injection were sparse. Fig. 6 shows photomicrographs of formalin-induced Fos-ir-positive cells in the spinal cord on the side ipsilateral to the site of the formalin injection. Numerous Fos-ir-positive cells were detected in laminae I–II and V–VI of the spinal cord in both vehicle-treated and 6-OHDA-treated rats at 2 h post formalin injection (Fig. 6A, B), followed by a decrease in the number of Fos-ir-positive cells at the 4-h time point (Fig. 6C, D). Table 1 shows the number of

Fos-ir-positive cells in laminae I–II and V–VI of vehicletreated and 6-OHDA-treated rats at the 2-h and 4-h time points (vehicle: 2 h, n = 7; 4 h, n = 7; 6-OHDA: 2 h, n = 8; 4 h, n = 9). A two-way ANOVA revealed that the numbers of Fos-ir-positive cells in laminae I–II or laminae V–VI of the ipsilateral spinal cord to the site of the formalin injection were significantly different from treatments [F(1, 27) = 7.41, p = 0.011; F(1, 27) = 14.36, p = 0.0008, respectively] and times [F(1, 27) = 38.63, p < 0.0001; F(1, 27) = 12.25, p = 0.0016, respectively]. However, there were no significant interactions between treatments and times [laminae I–II,

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laminae V–VI [F(1, 27) = 0.32, p = 0.58; F(1, 27) = 3.25, p = 0.083; F(1, 27) = 0.20, p = 0.66, respectively] of the contralateral spinal cord to the site of the formalin injection.

DISCUSSION

Fig. 6. Photomicrographs of transverse sections of L5 segments of the ipsilateral spinal cord to the side of formalin injection indicating the distribution of Fos-ir-positive cells induced by paw formalin injection in vehicle-treated rats (A and C) and 6-OHDA-treated rats (B and D). (A), (B) Fos-ir-positive cells 2 h post formalin injection. (C), (D) Fos-ir-positive cells 4 h post formalin injection. Scale bar = 300 lm.

F(1, 27) = 0.87, p = 0.36; laminae V–VI, F(1, 27) = 0.108, p = 0.75]. Further analysis with Bonferroni’s multiple comparison test showed that compared with the 2-h time points following the formalin injection, a significant decrease in the numbers of Fos-ir-positive cells was observed at the 4-h time points in laminae I–II and V–VI of vehicle-treated rats and in laminae I–II of 6-OHDA-treated rats (Vehicle: laminae I–II, p < 0.0001; laminae V–VI, p = 0.031; 6-OHDA: laminae I–II, p = 0.0011; laminae V–VI, p = 0.052). Fig. 7 shows differences in the numbers of Fos-ir-positive cells of the ipsilateral spinal cord to the site of the formalin injection between vehicle-treated and 6-OHDAtreated rats. Bonferroni’s multiple comparison test showed significant increases in the numbers of Fos-ir-positive cells in laminae V–VI of 6-OHDA-treated rats at 2 h (Fig. 7B; p = 0.0456), and in laminae I–II and laminae V–VI of 6-OHDA-treated rats at 4 h post formalin injection (Fig. 7A, B; p = 0.029 and 0.013, respectively). A two-way ANOVA revealed that treatment, time, and interactions between treatments and times did not significantly affect the numbers of Fos-ir-positive cells in laminae I–II [F(1, 27) = 0.67, p = 0.42; F(1, 27) = 0.009, p = 0.93; F(1, 27) = 0.59, p = 0.45, respectively] and

The current study is the first to report on the effects of neonatal dopamine depletion on nociceptive behavioral responses to various somatosensory stimuli, including mechanical, thermal, and chemical stimuli, during adolescence. This study demonstrates that neonatal dopamine depletion causes hyperalgesic behavioral response specifically to tonic chemical stimuli and motor hyperactivity during adolescence and that this abnormal response is associated with a long-lasting expression of the c-Fos protein in spinal neurons. In addition, these results suggest that the mechanism underlying the abnormal nociceptive behavior during adolescence, as a consequence of neonatal dopamine depletion, differs from that of motor hyperactivity induced by neonatal dopamine depletion. Several reports have shown that basal ganglia and the dopaminergic neuronal system are concerned with the modulation of somatosensory nociceptive inputs (Chudler and Dong, 1995; Coizet et al., 2006; Pelissier et al., 2006; Coizet et al., 2010). Ninety-two percent of the dopaminergic neurons in SN pars compacta exhibited a short-latency response to noxious stimulus, of which approximately 80% of the neurons exhibited inhibitory responses to the stimulus (Coizet et al., 2010). The administration of a D2R agonist into the striatum reportedly reduces the nociceptive behavioral response to a formalin injection (Magnusson and Fisher, 2000). Furthermore, tonic pain induced by chemical stimuli is more sensitive to dopaminergic agents compared with phasic thermal and mechanical pain (Pelissier et al., 2006; Camarasa et al., 2009). A hyperalgesic response to tonic chemical stimuli with increased nociceptive behavior during the end of phase 1 and late phase 2 in the formalin test has been reported using rats with dopamine depletion during adulthood (Tassorelli et al., 2007). Similarly, in the present study, adolescent rats with neonatal dopamine depletion exhibited hyperalgesic behavioral response to the formalin injection, showing increased nociceptive behavior during the interphase and late phase 2; however, abnormal responses to mechanical and thermal stimuli were not observed. Together with the results of previous studies (Pelissier et al., 2006; Tassorelli et al., 2007; Camarasa et al., 2009),

Table 1. Number of Fos-ir cells in laminae I–II and V–VI of the spinal cord Treatment

Time after formalin s.c.

Ipsilateral side Laminae I–II

Vehicle 6-OHDA

Mean ± 1 SEM. * p < 0.05. ** p < 0.01.

2h 4h 2h 4h

(n = 7) (n = 7) (n = 8) (n = 9)

**

55.4 ± 6.5 21.1 ± 2.7** 64.0 ± 5.2** 38.6 ± 4.1**

Contralateral side Laminae V–VI *

39.1 ± 6.3 17.8 ± 3.7* 58.3 ± 6.67 40.7 ± 4.97

Laminae I–II

Laminae V–VI

1.05 ± 0.46 1.38 ± 0.47 1.83 ± 0.53 1.41 ± 0.48

1.86 ± 0.65 1.00 ± 0.41 2.50 ± 0.88 1.07 ± 0.45

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Fig. 7. Comparison with number of Fos-ir-positive cells in laminae I–II and laminae V–VI of the ipsilateral spinal cord to the site of the formalin injection between vehicle-treated and 6-OHDA-treated rats. (A) The number of Fos-ir-positive cells in laminae I–II at 2 h and 4 h post formalin injection. (B) The number of Fos-ir-positive cells in laminae V–VI at 2 h and 4 h post formalin injection. n = 7, 7, 8 and 9 for vehicle-treated rats at 2 h, vehicle-treated rats at 4 h, 6-OHDA-treated rats at 2 h and 6-OHDA-treated rats at 4 h post formalin injection, respectively. ⁄p < 0.05.

these results suggest that the dopaminergic neural system plays a crucial role in pain processing, especially tonic chemical pain. However, whether the difference in the sensitivity of the dopaminergic neural system to the stimulus depends on the intensity or type of stimulus remains unclear, because withdrawal responses in the von-Frey and tail flick tests are measured as the threshold of the nociceptive response as opposed to the formalin test, in which the nociceptive response is induced by a suprathreshold stimulus. MAP is a psychostimulant that increases the extrace llular concentration of dopamine and noradrenaline by reversing the function of their corresp onding transporters (Sulzer et al., 2005). MAP and amphetamine also have a sedative effect on motor hyperactivity induced by neonatal dopamine depletion (Avale et al., 2004; Masuo et al., 2004). Rats with neonatal dopamine depletion in the present study also showed motor hyperactivity during adolescence. The administration of MAP did not ameliorate the hyperalgesic response to the formalin injection observed in rats with neonatal dopamine depletion, whereas analgesic effects on the nociceptive behavior were evident in vehicle-treated rats. The analgesic effect of MAP has been reported previously (Camarasa et al., 2009; Yamamotova´ et al., 2011), in which the effect was found to be dose dependent and was observed in the first and second phases of the formalin test (Camarasa et al., 2009), which is consistent with the results of the present study. The present study also revealed that rats with neonatal 6-OHDA treatment exhibited near-complete loss of TH-immunoreactive neurons in the midbrain but not LC, suggesting that the noradrenergic neural system was virtually intact. Taken together, these results suggest that the mechanism of the hyperalgesic response to the tonic chemical stimulus differs from that of motor hyperactivity induced by neonatal dopamine depletion, and that the dopaminergic, but not noradrenergic, neural system is implicated in the analgesic effect induced by MAP and the hyperalgesic behavioral response to the tonic chemical stimulus. However, to clearly distinguish those mechanisms, further studies are required to confirm whether the treatment of MAP showing analgesic effects on the nociceptive behavior of vehicletreated rats in the present study shows a sedative effect

on the motor hyperactivity of rats with neonatal dopamine depletion during adolescence. In the present study, the formalin injection into the hind paw was found to induce a long-lasting expression of the c-Fos protein at the spinal dorsal horn on the side ipsilateral to the formalin injection in rats with neonatal dopamine depletion (Fig. 7), indicating the hyperactivity of the spinal neurons in response to the tonic chemical stimulus. In rats with adult dopamine depletion, an increase in the numbers of Fos-ir-positive cells at the spinal dorsal horn on the side ipsilateral to the mechanical stimulation to the hind paw has been reported, and the reduced inhibitory serotonergic raphe projection to the spinal cord due to the reduction in the excitatory dopaminergic input to the raphe has been suggested (Reyes and Mitrofanis, 2008). The descending noradrenergic and serotonergic pathways to the spinal cord modulate the activity of nociceptive neurons (Jones, 1991; Millan, 2002; Pertovaara, 2006; Saade´ and Jabbur, 2008; Heinricher et al., 2009; Wu et al., 2010; Bardin, 2011). Descending dopaminergic controls of nociceptive transmission in the spinal and medullary dorsal horn have recently been reported (Lapirot et al., 2011; Taniguchi et al., 2011; Kawamoto et al., 2012). Using in vivo patch-clamp analysis, Taniguchi et al. (2011) reported that dopamine produces direct inhibitory effects on substantia gelatinosa neurons in the spinal cord in response to both noxious and innocuous stimulation of the skin. A reduction or facilitation of trigeminal nociceptive behavior induced by a formalin injection is observed with the local administration of a D2-like receptor agonist or antagonist, respectively, suggesting the existence of a tonic descending dopaminergic control of nociceptive transmission mediated via D2-like receptors in the medullary dorsal horn (Lapirot et al., 2011). The aforementioned results suggest that the hyperalgesic response to the formalin injection in the present study is attributable to the disinhibition of descending dopaminergic control caused by neonatal dopamine depletion. Since the direct dopaminergic projection from VTA exerts an inhibitory effect on electrical responses in the prefrontal cortex to mechanical noxious stimulation (Sogabe et al., 2013), the ascending dopaminergic projections should be considered to forward the future experiments.

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Previous studies have shown the effects of dopamine depletion during adulthood on nociceptive responses (Saade´ et al., 1997; Takeda et al., 2005; Tassorelli et al., 2007; Chudler and Lu, 2008); however, these results were not always consistent with those of the present study. Decreased latency of withdrawal responses to mechanical and/or thermal stimuli has been observed in the hemi-Parkinson rat model (Takeda et al., 2005; Chudler and Lu, 2008), as also has the hyperalgesic response to a formalin injection (Tassorelli et al., 2007; Chudler and Lu, 2008). The discrepancy between the results of the present and previous studies is attributable to the anatomical extent (unilateral or bilateral lesion in the dopaminergic neural system) and developmental period (adulthood or neonatal) of dopamine depletion. The mechanism underlying the paradoxical effect of dopamine depletion on behavioral response including motor activity has not yet been fully elucidated; however, the involvement of other neuronal systems such as serotonergic, GABAergic, and glutamatergic neuronal systems, is considered. Rats with neonatal 6-OHDA lesions exhibit decreased expressions of the GABA transporter and NMDA receptor 1 in the striatum at the age of 8 weeks (Masuo et al., 2004). Elevated 5-HT innervation of the striatum and increased expression of 5-HT2A receptors in striatal neurons are observed in rats with neonatal dopamine depletion (Snyder et al., 1986; Basura and Walker, 1999; Avale et al., 2004; Cunningham et al., 2005; Sivam et al., 2008). On the other hand, dopamine depletion during adulthood does not cause an apparent increase in 5-HT innervation (Breese et al., 1984; Stachowiak et al., 1984; Luthman et al., 1987). These differences in the effects of dopamine depletion on the development of neuronal elements as a consequence of the age of the 6-OHDA treatment are likely to contribute to the discrepancy observed in behavioral responses to nociceptive stimuli between the present and previous studies, in which animals with dopamine depletion during adulthood were used.

CONCLUSIONS In conclusion, the data from the present study indicate that neonatal dopamine depletion causes hyperalgesic behavioral response to chemical but not thermal and mechanical stimuli during adolescence, and that the hyperalgesic response is accompanied by neuronal hyperactivity in the spinal dorsal horn. The hyperalgesic response to the chemical stimulus is not ameliorated by pretreatment with MAP, which has sedative effects on motor hyperactivity induced by neonatal dopamine depletion, indicating that the mechanism underlying the hyperalgesic behavioral response was not always consistent with that of motor hyperactivity induced by neonatal dopamine depletion. These results suggest that the dopaminergic neural system, including the spinal neural circuit for nociceptive transmission, plays a crucial role in the development of tonic chemical pain, and that neonatal dopamine depletion causes long-lasting impairment in nociceptive behavioral function.

Acknowledgments—Authors thank Dr. Makoto Saji and Dr. Nobuyuki Suzuki for helpful comments and suggestions for our works. This work was supported by JSPA Grants-in-Aid for Scientific Research (23590721).

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(Accepted 5 January 2015) (Available online 12 January 2015)