Role of microglia in mechanical allodynia in the anterior cingulate cortex

Accepted Manuscript Role of microglia in mechanical allodynia in the anterior cingulate cortex Keisuke Miyamoto, Kazuhiko Kume, Masahiro Ohsawa PII:

S1347-8613(17)30088-9

DOI:

10.1016/j.jphs.2017.05.010

Reference:

JPHS 361

To appear in:

Journal of Pharmacological Science

Received Date: 10 April 2017 Revised Date:

12 May 2017

Accepted Date: 22 May 2017

Please cite this article as: Miyamoto K, Kume K, Ohsawa M, Role of microglia in mechanical allodynia in the anterior cingulate cortex, Journal of Pharmacological Science (2017), doi: 10.1016/ j.jphs.2017.05.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Role of microglia in mechanical allodynia in the anterior cingulate cortex Keisuke Miyamoto, Kazuhiko Kume, Masahiro Ohsawa Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya

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City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan * Corresponding Author,

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Masahiro Ohsawa, Ph.D.

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Associate Professor

Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, JAPAN

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Tel/Fax: +81-532-836-3410 e-mail: [email protected]

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Running Title: Cortex microglia and neuropathic pain

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Word count in the main body of the manuscript: 3995 words.

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ACCEPTED MANUSCRIPT Abstract Plastic changes that increase nociceptive transmission are observed in several brain

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regions under conditions of chronic pain. Synaptic plasticity in the anterior cingulate cortex (ACC) is particularly associated with neuropathic pain. Glial cells are considered candidates

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for the modulation of neural plastic changes in the central nervous system. In this study, we aimed to investigate the role of ACC glial cells in the development of neuropathic pain. First,

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we examined the expression of glial cells in the ACC of nerve-ligated mice. The expression of astrocytes and microglia was increased in the ACC of nerve-ligated mice, which was

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reversed by intracerebroventricular (i.c.v) treatment with the microglia inhibitor minocycline. Then, we examined the effect of minocycline on mechanical allodynia in nerve-ligated mice.

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I.c.v. and intra-ACC treatment with minocycline partially inhibited mechanical allodynia in the nerve-ligated mice. The expression of phosphorylated

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alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR1 subunit at Ser831, but not at Ser845, was increased in the ACC of the nerve-ligated mice compared to sham-operated mice, which was attenuated by minocycline administration. These results suggest that the activation of microglia in the ACC is involved in the development of hyperalgesia in mice with neuropathic pain. -2-

ACCEPTED MANUSCRIPT Keywords: Anterior cingulate cortex; minocycline; microglia; neuropathic pain; glutamate

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AMPA receptors

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ACCEPTED MANUSCRIPT 1. Introduction Chronic pain is a long-lasting symptom that persists after the healing of the tissue

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injury or lesion, and markedly lowers patients’ quality of life. Its main symptoms are hyperalgesia and allodynia. Among the several forms of chronic pain, neuropathic pain is one

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of the most refractory to treatment. Neuropathic pain results from the hyperexcitation of the sensory nerves that transmit nociceptive information (1). Once abnormal pain perception is

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established, symptoms are difficult to treat and afflict the patient. Although many studies have attempted to develop treatment strategies for neuropathic pain, there is currently no

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effective treatment.

Imaging studies using positron emission tomography (PET) have shown that

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nociceptive stimulation in patients with chronic pain activated the cingulate cortex (2,3). Magnetic Resonance Imaging (MRI) studies have also indicated that several brain regions are

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simultaneously activated during pain recognition (4). Pain perception consists of three distinct components, i.e. the sensory-discriminative, the affective-motivational, and the cognitive components. The sensory-discriminative component is encoded by the somatosensory cortical areas (S1 and S2), and recognizes the location, duration, and nature of noxious stimuli (2,3,5,6). The affective-motivational component is encoded by other cortical -4-

ACCEPTED MANUSCRIPT and limbic regions [anterior cingulate cortex (ACC), amygdala, and nucleus accumbens], and evokes unpleasant feelings and the motivation to escape from the noxious stimulus during

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pain perception (7,8). The brain regions involved in the affective-motivational component undergo substantial plastic changes in neuropathic pain (9). ACC and amygdala neuron

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hyperexcitability and pre- and post-synaptic long-term potentiation, in particular, have been demonstrated in several pain models (9,10). Therefore, enhanced neurotransmission in these

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regions might contribute to enhanced pain recognition in neuropathic pain. Central plasticity in the ACC has been associated with chronic pain. It has been

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reported that presynaptic release of glutamate and the function of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in ACC

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neurons are increased after nerve injury (11). Inhibition of the ACC glutamatergic system, especially inhibition of postsynaptic GluR1 accumulation, reversed neuropathic pain (12).

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However, the mechanisms underlying enhanced AMPA receptor function in the ACC of neuropathic pain are not fully understood. Recently, it was shown that neuroimmune alteration contributes to neural plasticity in neuropathic pain, especially in the spinal cord (13). The role of glial cells in neuropathic pain has been widely investigated in the spinal cord. Activation of spinal microglia and astrocytes is involved in the initiation and -5-

ACCEPTED MANUSCRIPT maintenance of neuropathic pain, respectively (14). Normalization of glial cells in the spinal cord of nerve-ligated mice ameliorated mechanical allodynia, suggesting that glial cells

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passively modulate nociceptive transmission through the modulation of excitatory neurotransmission in the spinal cord. In the ACC, role of activated astrocyte in negative

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emotion has been reported (15). Moreover, p38 mitogen-activated protein kinase that mainly expressed in microglia modulates pain-related negative emotion (16). Although direct

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evidence for the role of ACC astrocyte and microglia in sensory function has not been reported, pharmacological inhibition of astrocyte and microglia by gastrodin isolated from

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Chinese herb into the ACC attenuated the inflammation-induced spontaneous pain (17). Therefore, ACC microglia and astrocyte might be involved in the altered pain perception in

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neuropathic pain.

The functional changes in brain glial cells in neuropathic pain are not fully

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understood. In the spinal cord injury (SCI) model, microglia in the ventral posterolateral nucleus (VPL) of the thalamus are activated (18). The activation is observed in areas father from the injured region. This remote activation of microglia is caused by neural hyperexcitation after spinal cord injury. We considered the possibility that supraspinal remote activation of glial cells contributes to alternations in pain recognition in patients with -6-

ACCEPTED MANUSCRIPT neuropathic pain. In the present study, we aimed to examine the effect of centrally applied minocycline, a microglial inhibitor, on altered nociceptive processing in nerve-injured mice.

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Moreover, we examined molecular changes in the ACC under microglial inhibition.

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ACCEPTED MANUSCRIPT 2. Materials and Methods All animal experiment protocols used in this study were approved by the Animal

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Care and Use Committee of Nagoya City University, Japan and conducted in accordance with

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the guidelines of the National Institutes of Health and the Japanese Pharmacological Society.

2.1 Animals

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Male ddY mice, weighing 20–30 g, were used for every experiment. All animals were housed in a room, with six mice in each cage, maintained at 23 ± 2 ºC with an

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alternating 12 h- light-dark cycle. Animals had free access to food and water, and were used only once in all experiments. To induce unilateral hind-lib neuropathy, left sciatic nerve was

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without ligation.

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partially ligated as described previously (19). In sham-operated mice, the nerve was exposed

2.2 Assessment of mechanical threshold Mechanical allodynia was assessed by 50% likelihood of a paw withdrawal response (50% mechanical threshold). The 50% mechanical threshold was calculated by the up-down method as described by Dixon (20) using eight calibrated von Frey filaments -8-

ACCEPTED MANUSCRIPT (0.02-1.4g; Stoelting Wood Dale, IL). Calibrated filaments were applied vertically to the

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planter surface for 3–4 s. Quick paw lifting indicated a positive response.

2.3 Immunohistochemistry

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Seven days after nerve ligation, the animals were anesthetized with pentobarbital (60 mg/kg, i.p.) and intracardially perfused with 0.1 M phosphate-buffered saline (PBS)

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followed by 4% paraformaldehyde in PBS, and the whole brain was removed. ACC sections (50 µm) prepared with vivratome were incubated with 10% normal goat serum (NGS) for 1 h

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and then treated with rabbit polyclonal antibody against glial fibrillary acidic protein (GFAP) (1:8000; Dako, Carpinteria, CA) or Iba-1 (1:500; Wako Pure Chemical Industry Ltd., Kyoto,

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Japan) for 1 day at 4 ºC. The sections were then incubated with the Alexa 568 or 488-conjugated secondary antibody (1:2000; Thermo Fisher Scientific Inc., Suwannee, GA).

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The slides were then coverslipped with a fluorescence-mounting medium (Vector Laboratories, Burlingame, CA). The morphology of each section was analyzed using a laser scanning confocal microscope (LSM 700, CarlZeiss, Jena, Germany). Each area of Iba-1 or GFAP positive cells were assessed in the ACC. The Iba-1 or GFAP positive surface area was quantified using Image J software as % of the analyzed area. Analyzed area of each experiment -9-

ACCEPTED MANUSCRIPT was included ±5% from average. At least three independent experiments were performed.

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2.4 Western blot analysis The ACC was quickly removed following decapitation to evaluate protein

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expression. Equal protein amounts (20 µg) of samples were resolved in SDS-PAGE (4 % 20 %), and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA).

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The membrane was blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louise, MO) in Tris-buffered saline (pH 7.6) containing 0.05% Tween-20, and then incubated with rabbit

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polyclonal antibodies against GluR1 (1:1000; Upstate, Lake Placid, NY), Ser-831 GluR1 (1:1000 Upstate), Ser-845 GluR1 (1:1000; Chemicon International Inc., Temecula, CA), or

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β-actin (1:1000; Abcam) at 4 °C. The blots were visualized with enhanced chemiluminescence (SuperSignal West Dura Extended Duration Substrate; Thermo Fisher

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Scientific Inc.). The immunoblots were visualized using a LAS-3000 system (GE Healthcare Asia Co., Tokyo). The intensity of the band was analyzed and semi-quantified by computer-assisted densitometry using Image J. Each value was normalized by the respective value for β-actin as an internal control.

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ACCEPTED MANUSCRIPT 2.5 Microinjection to the anterior cingulate cortex in nerve-ligated mice Under pentobarbital anesthesia (60 mg/kg, intraperitoneally), the animals were

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fixed in a stereotaxic instrument. A 23-gauge guide cannula was unilaterally implanted above the ACC [A: +0.6 mm L: -0.3 mm H: +0.9 mm; (21)]. After two weeks from implantation of

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guide cannula, partial sciatic nerve ligated and sham-operated animals were prepared. A

30-gauge injection cannula was inserted into the guide cannula and minocycline (5 µg) was

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injected for 1 min (approximately 0.5 µL) using a microinjector pump (Eicom, Kyoto, Japan). The actual site of microinjection was confirmed by administrating 10% Evans blue with the

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same method used for minocycline or its vehicle on the final day.

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2.6 Drugs and intracerebroventricular (i.c.v.) injection The drug used in this study was minocycline (Sigma-Aldrich). Minocycline was

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dissolved in physiological saline (0.9% NaCl solution). Minocycline was administered intracerebroventriculary (i.c.v., 10 µg), or topically to the ACC (5 µg). I.c.v. administration was performed according to the method described by Haley and McCormick (22). Treatment with minocycline or vehicle was started 30 min before partial sciatic nerve ligation and was continued up to 6 days after nerve ligation. -11-

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2.7 Statistical analysis

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Data are expressed as mean ± SEM. Student’s t-test was used to examine differences between the two groups. The statistical significance of differences between multiple groups was

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0.05) were considered statistically significant.

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assessed with post-hoc Tukey tests. Differences in probability values of less than 0.05 (P <

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ACCEPTED MANUSCRIPT 3. Results 3.1 Activation of microglia in the anterior cingulate cortex (ACC) of nerve-ligated mice

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Seven days after nerve ligation, the expression of the microglial marker Iba-1-immunoreactivity (ir) (Fig. 1B) was increased unilaterally in the contralateral ACC of

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the nerve-ligated mice compared to that of the sham-operated mice (Fig. 1A). Daily repleated i.c.v. treatment with minocycline (10 µg) attenuated the increased expression of the Iba-1-ir

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(Fig. 1D) in the ACC of the nerve-ligated mice. In the sham-operated mice, minocycline treatment did not affect the expression of Iba-1-ir in the ACC (Fig. 1C). The intensity of

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Iba-1-ir expression was increased in the ACC of the nave-ligated mice, which was significantly attenuated by minocycline (Fig. 1E).

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(Fig. 1)

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3.2 Minocycline attenuates astroglial activation in the anterior cingulate cortex (ACC) of nerve-ligated mice

The expression of the astroglial marker GFAP-ir (Fig. 2B) was increased unilaterally in the contralateral ACC of the nerve-ligated mice compared to the ACC of the vehicle-treated sham-operated mice (Fig. 2A). Daily i.c.v. treatment with minocycline (10 -13-

ACCEPTED MANUSCRIPT µg) attenuated the increased expression of the GFAP-ir (Fig. 2D) in the ACC of the nerve-ligated mice. In the sham-operated mice, minocycline treatment did not affect the

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expression of GFAP-ir in the ACC (Fig. 2C). The intensity of GFAP-1-ir expression was

minocycline (Fig. 2E).

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(Fig. 2)

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increased in the ACC of the nave-ligated mice, which was significantly attenuated by

3.3 The effect of minocycline on hyperalgesia in nerve-ligated mice

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Daily intracerebroventricular (i.c.v.) administration of minocycline (10 µg) also improved mechanical hypersensitivity (Fig. 3A). In the contralateral paw, the mechanical

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threshold in nerve-ligated mice was not affected by i.c.v. treatment with minocycline. I.c.v. treatment with minocycline did not affect the mechanical nociceptive threshold in

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sham-operated mice (Fig. 3A).

Daily intra-ACC microinjection of minocycline (5 µg) after nerve ligation

improved mechanical hyperalgesia (Fig. 3B). The mechanical nociceptive threshold in the contralateral paw was not affected by intra-ACC treatment with minocycline. In the sham-operated mice, the mechanical nociceptive threshold in the contralateral and ipsilateral -14-

ACCEPTED MANUSCRIPT paw was not affected by intra-ACC treatment with minocycline. The microinjection site for

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(Fig. 3)

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each animal is shown in Fig. 3C.

3.4 Changes in glutamate AMPA receptors function in the ACC of nerve-ligated mice.

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A previous report has indicated the increased phosphorylation of glutamate AMPA receptor at Ser-831 in the ACC in a rat model of neuropathic pain (11). We investigated the

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effect of i.c.v.-administered minocycline on the expression of phosphorylated GluR1 in the ACC. The expression of GluR1 subunit phosphorylation at Ser831 was significantly

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increased in the ACC of the nerve-ligated mice compared to the ACC of the vehicle-treated sham-operated mice. (Fig. 4B). The increased phosphorylation of GluR1 subunit at Ser831

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was reversed by i.c.v. treatment with minocycline (Fig. 4B). In contrast, the expression of GluR1 subunit phosphorylation at Ser845 did not show significant changes in the ACC of the nerve-ligated mice. Moreover, the expression of the GluR1 subunit was not significantly increased in the ACC of the nerve-ligated mice compared to the ACC of the vehicle-treated sham-operated mice (Fig. 4D). -15-

ACCEPTED MANUSCRIPT (Fig. 4) It might be possible that minocycline attenuates phosphorylation of GluR1 subunit at Ser831

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through direct neural inhibition. We examined the effect of minocycline on N-methyl-D-aspartate (NMDA)-induced phosphorylation of GluR1 subunit in in vivo. I.c.v.

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treatment with N-methyl-D-aspartate (NMDA; 100 pmol) increased the expression of

phosphorylated GluR1 subunit at Ser831, but not Ser845, in the ACC (Fig. 5A, B and C).

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Acute treatment with minocycline (10 µg; i.c.v.) did not affected this increased phosphorylation of GluR1 subunit (Fig. 5B, C).

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(Fig. 5)

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ACCEPTED MANUSCRIPT 4. Discussion In the present study, we showed that minocycline exerts anti-allodynic effects

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through microglial inhibition in the anterior cingulate cortex (ACC), followed by astrocyte inhibition. This improvement might be resulted from the normalization of GluR1 at Ser831

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phosphorylation in the ACC of nerve-ligated mice.

Recent studies have illustrated the importance of the interaction between neurons

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and glial cells in neuropathic pain. Glial cells, especially astrocytes and microglia, interact with each other, and this interaction is apparent in in neuropathic pain. During the initial

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stage of neuropathic pain, there is a profound increase in microglia in the ipsilateral spinal dorsal horn of nerve-ligated mice (13). Pharmacological inhibition of microglia by

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minocycline attenuates mechanical hyperalgesia and allodynia in mice with neuropathic pain (14). In contrast to the spinal cord, it has been reported that supraspinal microglia are not

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activated in nerve-ligation (23). These results are contrary to ours. The reasons for this might be the use of different animal models (common peroneal nerve ligation vs. partial sciatic nerve ligation), differing experimental conditions (transgenic mice vs. immunohistochemistry), and experimental procedures (in vitro electrophysiology vs. in vivo behavioral study). Indeed, a recent study reported activation of microglia and astrocytes in -17-

ACCEPTED MANUSCRIPT the ACC after complete Freund’s adjuvant injection in mice (17). Although detail investigations are still required, it is likely that supraspinal, especially in the ACC, microglia

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might modulate excitatory neurotransmission in chronic pain conditions. Our results clearly indicated that the spatial activation patterns of microglia and

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that of astrocyte were different in the ACC of nerve-ligated mice. The exact reason for this different activation pattern is not clear. Microglial activation has been reported to precede the

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activation of astrocyte (14,24), Same temporal activation pattern was also observed in spinal cord injury model (25). This injury model also showed different spatial activation pattern of

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microglia (epi-center) and astrocyte (surrounded area). Based on these results, we speculate that the different activation patterns of microglia and astrocyte in the ACC resulted from the

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different temporal activation pattern of both glial cells. It is possible that minocycline directly attenuates the activity of astrocytes. A recent

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report indicated that cyclophposphamide-induced cystitis, which is modulated by spinal astrocytes, was not blocked by minocycline treatments (26). Therefore, it is more likely that minocycline-induced inhibition of astrocytes is due to the indirect effect of microglia inhibition. Previous report indicated that minocycline directly attenuates neuronal activity (27). It is possible that the effect of minocycline on phosphorylated GluR1 subunit is -18-

ACCEPTED MANUSCRIPT mediated by direct inhibition of neurons. Minocycline did not affect NMDA-induced increased phosphorylation of GluR1 subunit at Ser831in the ACC. Moreover, long-term

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potentiation after theta burst stimulation in the ACC was not affected by minocycline in normal mice brain slices (28). Therefore, it is possible that minocycline indirectly reduced

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phosphorylation of GluR1 subunit at Ser831.

Neuropathic pain model showed anxiety or depressive behaviors in 4-8 weeks after

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nerve ligation. Synaptic transmission in the ACC is enhanced on postsurgical days 7 (11). We observed only contralateral activation of microglia and astrocyte in the ACC of nerve ligated

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mice. Previous report indicated that bilateral activation of astrocyte was found 4 weeks after nerve ligation (29). Therefore, functional change of the ACC might be established before

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emergence of anxiety or depression in the neuropathic pain. The mechanisms of ACC microglial activation after sciatic nerve ligation are not

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fully understood. A previous study using a spinal cord injury model reported remote activation of thalamic microglia and astrocytes, which was mediated by sustained nociceptive input from the injured area (30). Since painful stimuli activate the ACC, it is possible that sustained excitatory input into the ACC might induce microglial activation. Multiple brain regions are involved in pain perception. A brain imaging study with -19-

ACCEPTED MANUSCRIPT humans showed that noxious stimuli activate the primary somatosensory cortex (S1), secondary somatosensory cortex (S2), ACC, insula, prefrontal cortex, thalamus, and

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cerebellum (31). It has been previously reported that synaptic plasticity in ACC neurons is causally related to neuropathic pain (32). A previous study illustrated that the induction of

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this synaptic plasticity requires the activation of glutamate NMDA receptors and L-type

voltage-gated calcium channels (33). In the present study, we indicated the involvement of

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ACC microglia in the induction of synaptic plasticity, as phosphorylation of the GluR1 subunit was attenuated by minocycline. Therefore, attenuation of GluR1 phosphorylation at

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Ser831 may be involved in the anti-allodynic effect of minocycline through the attenuation of synaptic plasticity in the ACC. It is also interesting that minocycline almost completely

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suppressed the activation of glial cells, whereas mechanical nociceptive threshold was partially reversed. This discrepancy between immunohistochemical and behavioral results

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might be due to another factors in the ACC might be involved in pain recognition. Indeed, protein kinase C zeta has been reported to be involved in the synaptic plasticity in the ACC of nerve ligation (10). It is not clear how GluR1 subunit was phosphorylated by the activated microglia. Activation of microglia regulates GluR1 phosphorylation in hippocampus in chronic -20-

ACCEPTED MANUSCRIPT unpredictable stress (34). Moreover, chemokine interleukin 8/CXCL8 induces phosphorylation of GluR1 subunit at Ser831 in neurons (35). Therefore, microglial mediators,

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especially interleukin 8, might be involved in phosphorylation of GluR1 subunit at Ser831 in the ACC of nerve-ligated mice.

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The present findings support the following hypothesis. Sustained excitatory input into the ACC after nerve ligation induces enhanced glutamatergic transmission. Activation of

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glutamate receptors increases calcium influx through the NMDA receptors. Increased intracellular calcium activates several protein kinases that phosphorylate numerous target

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intracellular signaling molecules, including GluR1. This hypothesis is also supported by a previous study showing that neurotransmission in the ACC was enhanced in a mouse model

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of neuropathic pain (36). The present study may deepen our understanding of the mechanisms

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involved in the neural hyperexcitability of these brain regions under neuropathic pain.

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ACCEPTED MANUSCRIPT Acknowledgements This study was supported by a Grant-in-Aid for Scientific research (B) Grant Number

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16H05460, and (C) Grant Number 25460724 (MO) from the Ministry of Education, Science,

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Sports, and Culture of Japan.

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ACCEPTED MANUSCRIPT Conflict of interest statement

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The authors declare no financial or other conflicts of interest related to this study.

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ACCEPTED MANUSCRIPT References (1)

Scholz J, Woolf CJ. Can we conquer pain? Nat Neurosci. 2002;5:1062–1067.

(2)

Jones AK, Brown WD, Friston KJ, Qi LY, Frackowiak RS. Cortical and subcortical localization of response to pain in man using positron emission tomography. Proceedings Biol Sci. 1991;244:39–44. Hsieh JC, Belfrage M, Stone-Elander S, Hansson P, Ingvar M. Central representation

RI PT

(3)

of chronic ongoing neuropathic pain studied by positron emission tomography. Pain. 1995;63:225–236. (4)

Bantick SJ, Wise RG, Ploghaus A, Clare S, Smith SM, Tracey I. Imaging how

attention modulates pain in humans using functional MRI. Brain. 2002;125:310–319. Qiu Y. Brain Processing of the Signals Ascending Through Unmyelinated C Fibers in

SC

(5)

Cortex. 2005;16:1289–1295. (6)

M AN U

Humans: An Event-Related Functional Magnetic Resonance Imaging Study. Cereb Kenshalo DR, Isensee O. Responses of primate SI cortical neurons to noxious stimuli. J Neurophysiol. 1983;50:1479–1496. (7)

Penfield W, Boldrey E. Somatic motor and sensory representation in the cerebral cortex of man as studied by electrical stimulation. Brain. 1937;60:389–443.

(8)

MacLean PD. Psychosomatic disease and the visceral brain; recent developments

(9)

TE D

bearing on the Papez theory of emotion. Psychosom Med. 11:338–353. Ikeda R, Takahashi Y, Inoue K, Kato F. NMDA receptor-independent synaptic plasticity in the central amygdala in the rat model of neuropathic pain. Pain. 2007;127:161–172.

Li X-Y, Ko H-G, Chen T, Descalzi G, Koga K, Wang H, et al. Alleviating neuropathic

EP

(10)

pain hypersensitivity by inhibiting PKMzeta in the anterior cingulate cortex. Science. (11)

AC C

2010;330:1400–1404.

Xu H, Wu L-J, Wang H, Zhang X, Vadakkan KI, Kim SS, et al. Presynaptic and

postsynaptic amplifications of neuropathic pain in the anterior cingulate cortex. J Neurosci. 2008;28:7445–7453.

(12)

Chen T, Wang W, Dong Y-L, Zhang M-M, Wang J, Koga K, et al. Postsynaptic insertion of AMPA receptor onto cortical pyramidal neurons in the anterior cingulate cortex after peripheral nerve injury. Mol Brain. 2014;7:76.

(13)

Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, et al. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature. 2003;424:778–783. -24-

ACCEPTED MANUSCRIPT (14)

Raghavendra V, Tanga F, Deleo JA. Inhibition of Microglial Activation Attenuates the Development but Not Existing Hypersensitivity in a Rat Model of Neuropathy. J Pharmacol Exp Ther. 2003;306:624–630.

(15)

Ikeda H, Mochizuki K, Murase K. Astrocytes are involved in long-term facilitation of neuronal excitation in the anterior cingulate cortex of mice with inflammatory pain.

(16)

RI PT

Pain. 2013;154:2836–2843. Cao H, Zang K-K, Han M, Zhao Z-Q, Wu G-C, Zhang Y-Q. Inhibition of p38

mitogen-activated protein kinase activation in the rostral anterior cingulate cortex

attenuates pain-related negative emotion in rats. Brain Res Bull. 2014;107:79–88. (17)

Sun T, Wang J, Li X, Li Y-J, Feng D, Shi W-L, et al. Gastrodin relieved complete

SC

Freund’s adjuvant-induced spontaneous pain by inhibiting inflammatory response. Int Immunopharmacol. 2016;41:66–73.

Zhao P, Waxman SG, Hains BC. Modulation of Thalamic Nociceptive Processing after

M AN U

(18)

Spinal Cord Injury through Remote Activation of Thalamic Microglia by Cysteine Cysteine Chemokine Ligand 21. J Neurosci. 2007;27:8893–8902. (19)

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;403:81–85. Dixon WJ. The up-and-down method for small samples. J Am Stat Assoc. 1965;60:967–978.

(21)

TE D

(20)

Paxinos G, Franklin KBJ. The Mouse Brain in Stereotaxic Coordinates. San Diego: Academic Press; 2001.

Haley TJ, McCormick WG. Pharmacological effects produced by intracerebral

EP

(22)

injection of drugs in the conscious mouse. Br J Pharmacol. 1957;12:12–15. Zhang F, Vadakkan KI, Kim SS, Wu L-J, Shang Y, Zhuo M. Selective activation of

AC C

(23)

microglia in spinal cord but not higher cortical regions following nerve injury in adult mouse. Mol Pain. 2008;4:15.

(24)

Raghavendra V, Tanga F, Rutkowski MD, DeLeo JA. Anti-hyperalgesic and morphine-sparing actions of propentofylline following peripheral nerve injury in rats: mechanistic implications of spinal glia and proinflammatory cytokines. Pain. 2003;104:655–664.

(25)

Okada S, Nakamura M, Katoh H, Miyao T, Shimazaki T, Ishii K, et al. Conditional ablation of Stat3 or Socs3 discloses a dual role for reactive astrocytes after spinal cord injury. Nat Med. 2006;12:829–834. -25-

ACCEPTED MANUSCRIPT (26)

Liu B, Su M, Tang S, Zhou X, Zhan H, Yang F, et al. Spinal astrocytic activation contributes to mechanical allodynia in a rat model of cyclophosphamide-induced cystitis. Mol Pain. 2016;12.

(27)

González JC, Egea J, Del Carmen Godino M, Fernandez-Gomez FJ, Sánchez-Prieto J, Gandía L, et al. Neuroprotectant minocycline depresses glutamatergic

RI PT

neurotransmission and Ca(2+) signalling in hippocampal neurons. Eur J Neurosci. 2007;26:2481–2495. (28)

Song Q, Liu M-G, Zhuo M. Minocycline does not affect long-term potentiation in the anterior cingulate cortex of normal adult mice. Mol Pain. 2015;11:25.

(29)

Narita M, Kuzumaki N, Narita M, Kaneko C, Hareyama N, Miyatake M, et al. Chronic

SC

pain-induced emotional dysfunction is associated with astrogliosis due to cortical delta-opioid receptor dysfunction. J Neurochem. 2006;97:1369–1378. Daou I, Tuttle AH, Longo G, Wieskopf JS, Bonin RP, Ase AR, et al. Remote

M AN U

(30)

optogenetic activation and sensitization of pain pathways in freely moving mice. J Neurosci. 2013;33:18631–18640. (31)

Bushnell MC, Ceko M, Low L a. Cognitive and emotional control of pain and its disruption in chronic pain. Nat Rev Neurosci. 2013;14:502–511.

(32)

Wei F, Zhuo M. Potentiation of sensory responses in the anterior cingulate cortex

(33)

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following digit amputation in the anaesthetised rat. J Physiol. 2001;532:823–833. Liauw J, Wu L-J, Zhuo M. Calcium-stimulated adenylyl cyclases required for long-term potentiation in the anterior cingulate cortex. J Neurophysiol. 2005;94:878– 882.

Liu M, Li J, Dai P, Zhao F, Zheng G, Jing J, et al. Microglia activation regulates GluR1

EP

(34)

phosphorylation in chronic unpredictable stress-induced cognitive dysfunction. Stress. (35)

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2015;18:96–106.

Catalano M, Trettel F, Cipriani R, Lauro C, Sobrero F, Eusebi F, et al. Chemokine CXCL8 modulates GluR1 phosphorylation. J Neuroimmunol. 2008;198:75–81.

(36)

Koga K, Descalzi G, Chen T, Ko H-G, Lu J, Li S, et al. Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and

Chronic Pain. Neuron. 2015;85:377–389.

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ACCEPTED MANUSCRIPT Figure legends Fig. 1. Expression of Iba-1-immunoreactivity (ir) in the anterior cingulate cortex (ACC) 7

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days after nerve ligation. Daily treatment with minocycline (10 µg, i.c.v.; C, D) or its vehicle (saline, i.c.v., A, B) was started 1 hour before nerve ligation and was continued

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until 6 days after nerve ligation. Coronal sections of the ACC were prepared from vehicle-treated sham-operated (Sham-Saline; A), nerve-ligated (PNL-Saline; B),

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minocycline-treated sham-operated (Sham-Mino; C), and nerve-ligated (PNL-Mino; D) mice. The ACC was removed from sham-operated and nerve-ligated mice 7 days

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post-surgery. Three separate samples were used in this study. Scale bar = 100 µm. E: Semi-quantitative analysis of the area of Iba-1-ir in ACC of neuropathic and

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sham-operated mice. The area of Iba-1-ir in the ACC was quantified using Image J software. Each column represents the mean ± SEM from 3 independent experiments.

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**P<0.01 vs sham-operated group. ##P<0.01 vs. respective saline-treated group.

Fig. 2. Expression of GFAP-immunoreactivity (ir) in the ACC 7 days after nerve ligation. Treatment with minocycline (10 µg, i.c.v.; C, D) or its vehicle (saline, i.c.v., A, B) was started 1 hour before nerve ligation and was continued until 6 days after nerve ligation. -27-

ACCEPTED MANUSCRIPT Coronal sections of the anterior cingulate cortex were prepared from vehicle-treated sham-operated (Sham-Saline; A), nerve-ligated (PNL-Saline; B), minocycline-treated

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sham-operated (Sham-Mino; C), and nerve-ligated (PNL-Mino; D) mice. The ACC was removed from sham-operated and nerve-ligated mice 7 days post-surgery. Three

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separate samples were used in this study. Scale bar = 200 µm. E: Semi-quantitative analysis of the area of GFAP-ir in ACC of neuropathic and sham-operated mice. The

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area of GFAP-ir in the ACC was quantified using Image J software. Each column represents the mean ± SEM from 3 independent experiments. **P<0.01 vs

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sham-operated group. ##P<0.01 vs. respective saline-treated group.

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Fig. 3. The effect of minocycline on mechanical hyperalgesia in nerve-ligated mice. Intracerebroventricular (i.c.v; A) or intra-anterior cingulate cortex (ACC; B) treatment

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with minocycline was started 1 hour before nerve ligation and was continued until 6 days after nerve ligation. Mice was assessed by the von Frey test. Each point represents the mean ± SEM of 8 mice (A) or 5 mice (B). *P < 0.05 and **P < 0.01 vs. before nerve ligation. #P < 0.05 vs. respective vehicle-treated group. Panel (C) indicates the injection sites of minocycline in the ACC. -28-

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Fig. 4. Expression of phosphorylated AMPA receptor subunit GluR1 at serine 831 (B), serine

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845 (B), and total GluR1 in the anterior cingulate cortex (ACC) of nerve-ligated mice. (A) The representative immunoblots showing the amount of pGluR1 (ser831), pGluR1

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(ser845), GluR1, and β-actin (internal control) from the ACC. The immunoblots from the same antibody obtained from the same membrane. The intensity of all GluR1

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immunoblots was normalized by β-actin. Each column represents the mean ± SEM of four separate experiments. *P < 0.05 vs. vehicle-treated sham-operated mice. #P < 0.05

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vs. respective vehicle-treated group.

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Fig. 5. The effect of minocycline on NMDA-induced phosphorylation of GluR1 subunit at serine 831 (B), and serine 845 (C). Minocycline (10µg, i.c.v.) was pretreated 30

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minutes before the NMDA (100 pmol, i.c.v.) treatment. The ACC was removed quickly 2 hours after treatment of NMDA. (A) The representative immunoblots showing the amount of pGluR1 (ser831), pGluR1 (ser845), and each of β-actin (internal control). The intensity of all GluR1 immunoblots was normalized by β-actin. Each column represents the mean ± SEM of four separate experiments. *P < 0.05 vs. -29-

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saline-pretreated saline group.

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Iba-1 / DAPI

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50% Mechanical Threshold (g)

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Bregma 0.62mm Bregma 0.98mm Bregma 1.18mm

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