Presentation of noise during acute restraint stress attenuates expression of immediate early genes and arginine vasopressin in the hypothalamic paraventricular nucleus but not corticosterone secretion in rats.

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Presentation of noise during acute restraint stress attenuates expression of immediate early genes and arginine vasopressin in the hypothalamic paraventricular nucleus but not corticosterone secretion in rats

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Koji Sugimoto a,1 , Hideki Ohmomo b,2 , Fumihiro Shutoh a,c , Haruo Nogami a,c , Setsuji Hisano a,c,∗ a

Laboratory of Neuroendocrinology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan Division of Biomedical Information Analysis, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan Q2 c Laboratory of Neuroendocrinology, Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan b

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Article history: Received 19 September 2014 Received in revised form 12 November 2014 Accepted 28 November 2014 Available online xxx

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Keywords: Acoustic stimulus White noise 22 c-Fos 23 JunB 24 Arginine vasopressin 25 26 Q4 Corticotropin-releasing hormone Corticosterone 27 Paraventricular nucleus 28 20 21

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The present study investigated the effect of acoustic stimulation on the activation of the hypothalamicpituitary-adrenal (HPA) axis in rats submitted to acute restraint stress, through semi-quantitative histochemical analysis of expression of immediate early gene products (c-Fos, JunB and phosphorylated c-Jun) and arginine vasopressin (AVP) hnRNA in the paraventricular nucleus (PVN). Simultaneous presentation of white or pink noise with restraint resulted in a significant attenuation of stress-induced c-Fos and JunB expression in the dorsal body of dorsal medial parvicellular subdivision (mpdd) of the PVN, as compared with restraint without noise. However, this presentation did not change phosphorylation of c-Jun and the plasma corticosterone level. Moreover, white noise presentation during restraint led to a reduction in the number of c-Fos- or JunB-expressing corticotropin-releasing hormone (CRH) neurons and the number of neurons expressing AVP hnRNA in the mpdd. Dual-histochemical labeling revealed co-expression of c-Fos and JunB, as well as JunB and AVP hnRNA in mpdd neurons. These data suggest that acoustic stimuli have an attenuation effect on the restraint-induced activation of neuroendocrine CRH neurons, resulting in the reduction in AVP production as an adaptation of HPA axis to repeated stress. © 2014 Published by Elsevier Ireland Ltd.

1. Introduction As the final common pathway in the hypothalamic-pituitary-adrenal (HPA)

stress response, axis drives the

Abbreviations: HPA, hypothalamic-pituitary-adrenal; CRH, corticotropinreleasing hormone; PVN, paraventricular nucleus; ACTH, adrenocorticotropic hormone; CORT, corticosterone; AVP, arginine-vasopressin; IEG, immediate early gene; pc-Jun, phosphorylated c-Jun; AP1, activator protein 1; hnRNA, heterogeneous nuclear RNA; mRNA, messenger RNA; mpd, dorsal medial parvicellular part; mpdd, dorsal body of the mpd; mpdv, ventral tail of the mpd; dp, dorsal parvicellular part; mpv, ventral medial parvicellular part; pv, periventricular parvicellular part; pm, posterior magnocellular part. Q3 ∗ Corresponding author at: Laboratory of Neuroendocrinology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan. Tel.: +81 29 853 3100; fax: +81 29 853 3100. E-mail address: [email protected] (S. Hisano). 1 Present address: Laboratory of Advanced Research D, 1-1-1 Tennodai, Tsukuba-shi, Ibaraki 305-8577, Japan. 2 Present address: 2-1-1 Nishitokuta, Yahaba-cho, Shiwa, Iwate-ken 028-3694, Japan.

neuroendocrine response necessary for survival of organisms to cope with physical and psychological stressors. The neuroendocrine response begins with excitation of corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN), Q5 followed by the adrenocorticotropic hormone (ACTH) release from the anterior pituitary gland, and finally ends with the release of the adrenal glucocorticoid, corticosterone (CORT) in the rodents (Watts, 2005). Interestingly, recent studies have reported attenuation effects of an olfactory (Ito et al., 2009) or a gustatory stimulus (Martin and Timofeeva, 2010) on the HPA response to acute restraint stress. As for an acoustic stimulus, however, no study has addressed that effect on the stress response, although one reported no effect of loud noise as a novel heterotypic stressor in the habituation to repeated restraint stress (Masini et al., 2012). In exploring for the possible effect of acoustic perception on restraint stress response, the most critical choice is that of the stimulus suitable for evaluation of the HPA response. In this sense, white noise may be appropriate, because it has been widely used in previous studies (Day et al., 2005; Spiga et al., 2009). The physiological

http://dx.doi.org/10.1016/j.neures.2014.11.010 0168-0102/© 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Sugimoto, K., et al., Presentation of noise during acute restraint stress attenuates expression of immediate early genes and arginine vasopressin in the hypothalamic paraventricular nucleus but not corticosterone secretion in rats. Neurosci. Res. (2014), http://dx.doi.org/10.1016/j.neures.2014.11.010

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effect of white noise in rats seems to depend on its intensity in presentation. A loud (above 90 dB) white noise is often employed as a stressor to stimulate the HPA axis (Burow et al., 2005; Masini et al., 54 2012), while white noise at 80 dB has an overshadowing effect, 55 56Q6 possibly an anxiolytic effect (Zelikowsky et al., 2010). Also of importance is what indicator is used to assess the HPA 57 response, especially the magnitude of neuroendocrine CRH neu58 ron excitation. Within the PVN, CRH neurons exist principally in 59 the dorsal medial parvicellular part (mpd), and produce arginine 60 61Q7 vasopressin (AVP) as an ACTH co-secretagogue in the HPA axis (Engelman et al., 2004; Piet and Manzoni, 2011). When stressed, 62 the neurons release these hormones and initiate expression of crh 63 and avp genes, as well as immediate early genes (IEG) such as c-fos, 64 junB and c-jun (Imaki et al., 1996; Kovács and Sawchenko, 1996; 65 Kovács et al., 1998). In hormone production, CRH heterogeneous 66 nuclear RNA (hnRNA) is more rapidly (within 30-min post-stress) 67 expressed (Liu et al., 2011), whereas AVP hnRNA expression is 68 delayed until 120 min after stress, concomitantly with or later than 69 IEG protein expression (Kovács and Sawchenko, 1996). Although 70 c-Fos does not seem to be a genuine transcription factor of crh gene, 71 it has often been used as a cell marker that allows us to assess stress72 induced CRH neuron excitation (Imaki et al., 1996; Stamp and 73 Herbert, 1999; Chowdhury et al., 2000; Viau and Sawchenko, 2002; 74 Girotti et al., 2006). Thus, the aim of the present study is to investi75 gate whether or not acoustic stimulus presentation affects the HPA 76 response in rats submitted to acute restraint stress, through semi77 quantitative histochemical analysis for IEG and AVP expression as 78 well as an assay of the plasma CORT level. 79 52 53

of each noise when measured with a sound level meter (SM-325, As One, Osaka, Japan). White noise was generated using free software KSK Funcgen. Pink and blue noise were made from white noise using free software (SoundEngine Free). These noise included components of 20 Hz–20 kHz frequency bands. We examined the following five experimental groups: (1) restraint stress without artificial noise (S group); (2) restraint stress plus white noise (W group); (3) restraint stress plus pink noise (P group); (4) restraint stress plus blue noise (B group); and (5) intact, namely without both restraint stress and artificial noise (I group). Rats of all but the groups except the I group were left in the box for 30 min with or without noise presentation, and then brought back to their home cages. The rats of I group were continuously housed in home cages before tissue sampling. 2.3. Tissue preparation Two hours after the onset of restraint (the point in time when rats were placed in the box) of the S, W, P and B groups, as well as immediately before sacrifice of the I group, the rats were deeply anesthetized with sodium pentobarbital (75 mg/kg, i.p.) and transcardially perfused with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.5). The hypothalamus was dissected out, postfixed in the same fixative overnight, and immersed in 30% sucrose in PB. The tissues were embedded and frozen, then cut on a cryostat (Cryostat HM560 MV, Carl Zeiss, Oberkochen, Germany) into 10-␮m thick coronal serial PVN sections, and stored at −80 ◦ C until analysis.

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2.4. Immunostaining

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In the analysis of IEG expression, c-Fos and JunB were examined in all groups (n = 5 each), while phosphorylation of c-Jun was studied in the S (n = 5), W (n = 5) and I (n = 4) groups. Sections were microwaved for 7 min, treated with 0.3% H2 O2 in 20 mM phosphate-buffered saline (PBS, pH 7.5) for 30 min, and incubated in blocking reagent (20 mM PBS, 0.1% NaN3 , and 10% normal goat or horse serum) for 1 h. Immunostaining for c-Fos, JunB and phosphorylated c-Jun (pc-Jun) was performed with avidin-biotinperoxidase complex (ABC) method as follows: the sections were incubated in either an anti-c-Fos antibody (1:7500, Poly6414, Biolegend, San Diego, CA) (24 h, 4 ◦ C), an anti-JunB antibody (1:1000, sc-8051, Santa Cruz, Texas) (72 h, 4 ◦ C), or an anti-pc-Jun (Ser63) antibody (1:100, #9261, Cell Signaling Technology, Danvers, MA) (24 h, 4 ◦ C); then in either biotinylated (b-) goat anti-rabbit or horse anti-mouse IgGs (1:200, Vector Laboratories, Burlingame, CA) (1 h, 36 ◦ C); and finally in ABC (Vectastain® ABC Elite Kit, Vector) (1 h, 36 ◦ C). Immunoreaction was visualized using 3,3 diaminobenzidine tetrahydrochloride (DAB, Nacalai, Kyoto, Japan) in 20 mM PBS containing 0.003% H2 O2 (8 min, 37 ◦ C). After PBS washing, the sections were coverslipped with 50% glycerol, and photographed with an AxioCam MRc5 equipped on an Axioskop2 plus microscope (Carl Zeiss). For dual immunostaining of c-Fos and CRH in the S and W groups (n = 5 each), c-Fos-stained sections were treated sequentially in H2 O2 , blocking reagent and an anti-CRH antibody (1:15,000, T-4037, Peninsula Laboratories, San Carlos, CA) (24 h, 4 ◦ C). The sections were incubated with b-goat anti-rabbit IgG (1:200, Vector) and ABC (Vector) (1 h, 36 ◦ C each). CRH immunoreaction was visualized using Vector® SG Substrate Kit (Takahashi et al., 2011). For immunofluorescence staining, sections of S group (n = 4) were treated as follows: (1) the c-Fos antibody (1:5000) (24 h, 4 ◦ C); (2) b-secondary antibody (1:200, Vector); (3) the JunB antibody (1:1000) (72 h, 4 ◦ C); (4) streptavidin conjugated with a mixture of Alexa Fluor® 555 (1:200, Invitrogen, Carlsbad, CA) and Alexa Fluor® 633 goat anti-mouse IgG (1:200, Invitrogen) in 20 mM PBS with 10%

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All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This study was conducted with the approval of the Animal Experiment Committee of the University of Tsukuba, and subjected to the regulations of the university requirements regarding the care and use of laboratory animals for experimental procedures. All efforts were made to minimize the number of animals used and their suffering. Adult male Sprague-Dawley strain rats were purchased (Nippon Clea, Tokyo, Japan), weighing 290–310 g at the time of the experiment, and maintained under a 12-h light/dark cycle (light on 07:30) at 25 ◦ C with food and water ad libitum. After they were obtained, the rats were acclimated by handling everyday until the experiment day, and housed individually for 4 days before experiment.

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2.2. Restraint stress and presentation of acoustic stimuli

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2.2.1. Stress paradigm Each rat was placed into a transparent plastic tube (5 cm in inner diameter, 25 cm in length), having a front hole 1.3-cm in diameter to avoid accidental injury to the nose, and several lateral sound through-holes 0.5-cm in diameter. Immediately after introducing a rat into the tube, the rat was placed in a soundproof box (50 cm × 50 cm × 40 cm) interiorly at 1000 lux with background noise (50 dB). 2.2.2. Presentation of stimuli Simultaneously with restraint, rats in the box were presented with either white, pink or blue noise played back with an audio player (20 Hz–20 kHz, XU-D400 MK II, JVC Kenwood Corporation, Japan), through two speakers (ASP-1000N Speaker, OHM Electric Inc., Japan) placed 36 cm above the box floor. The total sound pressure level within the box was always 70 dB during presentation


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goat serum; and (5) 4 ,6-diamidine-2 -phenylindole dihydrochloride (DAPI) (1 ␮g/mL, Roche Diagnostic, Indianapolis, IN) in 20 mM PBS (15 min, 36 ◦ C). The sections were photographed under a laser scanning microscope (LSM 510, Carl Zeiss). All positive staining for c-Fos, JunB, pc-Jun and CRH was abolished when incubated with the primary antibodies preabsorbed with the respective immunogen: c-Fos (167 ␮g/mL c-Fos peptide kindly supplied by Biolegend), JunB (100 ␮g/mL blocking peptide, sc-8051 P, Santa Cruz), pc-Jun (100 ␮g/mL blocking peptide, sc-7980 P, Santa Cruz), and CRH (10 ␮M full length rat peptide, 4136-s, Peptide Institute, Osaka, Japan).

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Digoxigenin (DIG)-labeled antisense and sense cRNA probes were transcribed with cDNA encoding rat CRH exon2 (417 bp) or rat AVP intron1 (628 bp). Total RNAs of the fresh rat diencephalon prepared with the method of Chomczynski and Sacchi (1987) were reversed transcribed into the cDNAs. RT-PCR was carried out using LA-taq DNA polymerase (Takara, Shiga, Japan) with CRH exon2 primers (forward: 5 -ccgttgaatttcttgcaacc-3 , reverse: 5 -gttgctgtgagcttgctgag-3 ) or AVP intron1 primers (forward: 5 tagatgggtttcccagaacg-3 , reverse: 5 -cgccaacctattatgcccta-3 ), and then the synthesized CRH or AVP cDNA fragment was inserted into pCR® II vector (Invitrogen). This plasmid was linearized by EcoRV or BamH I (Takara), and used as a template for in vitro transcription using SP6 or T7 polymerase (Takara).

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After c-Fos or JunB immunostaining, sections of the S and W groups (n = 5 each) were treated with 0.1 M triethanolamine containing 0.25% acetic anhydride, washed, and then incubated in pre-hybridization buffer (50% formamide, 5% SDS and 5× SSPE) (1 h, 60 ◦ C). The sections were hybridized in hybridization solution (pre-hybridization buffer containing 1 ␮g/mL CRH cRNA probe and 1 mg/mL yeast tRNA) (18 h, 60 ◦ C). After hybridization, the sections were washed in post-hybridization buffer (50% formamide, 1.5 M NaCl and 15 mM sodium citrate) (1.5 h, 60 ◦ C), and maleic acid buffer (pH 7.5; 0.1 M maleic acid, 50 mM NaCl and 0.1% Tween 20). DIG-labeled cRNA hybrids were visualized as follows: the sections were incubated in 2% blocking reagent (Roche) containing 10% sheep serum, and in an alkaline phosphatase (AP)-labeled anti-DIG antibody (1:1000, Roche) (18 h, 4 ◦ C). After washing with Tris–buffer (0.1 M Tris–HCl, 10 mM NaCl, 0.5 M MgCl2 and 0.1% Tween 20), the sections were incubated in NBT/BCIP mixture (0.3375 mg/mL nitro-blue-tetrazolium-chloride (Roche), 0.175 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate (Roche) and 10% polyvinyl alcohol in 0.1 M Tris–buffer).

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Sections of the S (n = 9), W (n = 10) and I (n = 9) groups were re-fixed in paraformaldehyde and processed in the posthybridization step described above. After peroxidase and endogenous biotin activities were inactivated, the sections were incubated with Tris–NaCl-blocking buffer (0.5% blocking reagent (TSA kit, PerkinElmer, Waltham, MA) in 0.1 M TBS; pH 7.5), and then with an anti-DIG antibody conjugated with horseradish peroxidase (1:500, Roche) (20 h, 4 ◦ C). To detect AVP hn cRNA hybrids, the sections were successively processed in tyramide signal amplification (Bobrow et al., 1989) at room temperature as follows: (1) b-tyramide (1:100, PerkinElmer) in 0.1 M TBS (pH 7.5) containing 0.001% H2 O2 (5 min); (2) streptavidin conjugated with horseradish peroxidase (1:5000, Merck Millipore) (30 min); (3) b-tyramide (1:50) in the TBS containing H2 O2 (5 min); and (4) streptavidin

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conjugated with AP (1:50,000, Roche) (30 min). AP activity was visualized using NBT/BCIP mixture. For a combination of JunB immunostaining and AVP hnRNA in situ hybridization, after treatments with 20% acetic acid (30 min, 4 ◦ C) followed by blocking reagent, sections of the S group were incubated in order as follows: the JunB antibody (72 h, 4 ◦ C); b-horse anti-mouse IgG (1:200, Vector); and ABC-AP solution (Vectastain® ABC-AP Kit, Vector). JunB immunoreaction was visualized using a Vector® Red Substrate Kit (Vector). The sections mounted with 50% glycerol were coverslipped, and photographed. After removal of covers, the sections were treated again in 20% acetic acid (30 min, 4 ◦ C), and hybridized for AVP hnRNA as described above. 2.8. Plasma CORT assay We examined the S and I groups (n = 5 each) to find the effect of restraint stress, and all groups (n = 8 each) to find the effect of noise presentation. Immediately after a 30-min restraint stress for S, W, P and B groups, as well as immediately after picked up from their cage for the I group, rats were decapitated and trunk blood was collected onto centrifugation tubes containing 1 mg/mL EDTA·2Na. Blood samples were centrifuged at 3000 × g (10 min, 4 ◦ C), and then the plasma was stored at −80 ◦ C until assay. Plasma CORT levels in the duplicated blood samples were measured by enzyme-linked immunosorbent assay (ELISA) method (Corticosterone ELISA Kit, Assaypro, St. Charles, MO) according to the protocol of the testing kit, and the absorbance was recorded in a microplate reader (MTP310, Corona Electric Co., Ibaraki, Japan). 2.9. Semi-quantification procedure Parcellation of the PVN subdivisions was determined according to the atlas of Swanson (1998/1999). At the frontal plane of PVN corresponding to level 26 in the atlas, we analyzed the following six subdivisions: dorsal body (mpdd) and ventral tail (mpdv) of the mpd, a dorsal parvicellular part (dp), ventral medial parvicellular part (mpv), periventricular parvicellular part (pv), and posterior magnocellular part (pm). Using software (Photoshop® CS5, Adobe Systems, San Jose, CA), lines of subdivision boundaries were drawn by tracing nissl-stained sections according to previ- Q8 ous studies (Swanson and Simmons, 1987; Viau and Sawchenko, 2002), and were superimposed onto images of immunostaining or in situ hybridization in adjacent sections. To standardize the intensity of the immunostaining or AVP hnRNA signal, superimposed images were converted to grayscale images. The intensity in the ependyma, defined as the background, was subtracted from the grayscale image of each section using Photoshop tone-curve tool. Using Image J (National Institutes of Health), the threshold range was set to 0–254 for immunostaining and to 0–250 for in situ hybridization images. The pixel-size of analyzed particles was set to 100- and 20-infinity for immunostaining and in situ hybiridization images, respectively, and nuclei expressing IEG or AVP hnRNA were automatically counted in each subdivision. In the analysis of dual-labeled sections, CRH-stained perikarya were counted through the separation of a dual-color image into two single-color images using Color Deconvolution tool. Overlaps of positive nuclei were microscopically checked visually and the number was corrected appropriately. 2.10. Statistical analysis Data were expressed by mean ± SEM and analyzed for statistical significance by one-way ANOVA followed by Dunnett’s T3 post hoc test or Welch t-test. Significant differences were defined at p < 0.05.


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Fig. 1. Immunostaining and semi-quantification of c-Fos (A–C) and JunB (D–F) expression in the PVN of I group (A and D), S group (B and E) and W group (C and F). c-Fos (B) and JunB (E) are expressed 2 h after the start of restraint (S group), and are attenuated by simultaneous white noise presentation with restraint (W group; C and F). Scale bar = 200 ␮m. The number of c-Fos or JunB-expressing nuclei is larger in the mpdd, mpdv and pm of the S group than in the I group (G), and smaller only in the mpdd of the W and P groups than in the S group (G). There was no significant difference between the B and the S groups. Data were expressed by mean ± SEM and analyzed for Welch t-test and one-way ANOVA followed by Dunnett’s T3 post hoc test, **p < 0.01 vs. I group, † p < 0.05 vs. S group, n = 5 per group. mpdd, dorsal body of the dorsal medial parvicellular part; mpdv, ventral tail of the dorsal medial parvicellular part; mpv, ventral medial parvicellular part; dp, dorsal parvicellular part; pm, posterior magnocellular part; pv, parvicellular part. The detailed explanation of the groups (I, S, W, P, B) is described in Section 2.

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3. Results 3.1. c-Fos and JunB expression in PVN and plasma CORT level after restraint Neurons expressing c-Fos were sparse in the PVN of I group (Fig. 1A). In contrast, c-Fos was expressed principally in the parvicellular PVN of S group (Fig. 1B). JunB expression was found only a little in the I group (Fig. 1D), but was easily observable in the S group, especially in the mpd (Fig. 1E). Semi-quantification revealed that more c-Fos-expressing nuclei exist (p < 0.01) in the mpdd, mpdv and pm (Fig. 1G), and in the mpv, dp and pv (data not shown) of the S group than in these subdivisions of the I group. Also, significantly more (p < 0.01) JunB-expressing nuclei were found in

the same subdivisions (Fig. 1G) of the S group than in the I group (Fig. 1G). The plasma CORT level was significantly higher (p < 0.01) in S group (288.1 ± 14.8 ng/ml, n = 5) than in I group (18.1 ± 2.3 ng/ml, n = 5). 3.2. The effect of acoustic stimuli presentation on stress-induced c-Fos and JunB expression and the elevation of plasma CORT level Immunostaining suggested that both c-Fos (Fig. 1B and C) and JunB (Fig. 1E and F) expression is less intense in W group than in S group. Semi-quantification showed that there is a significant difference only in the mpdd (c-Fos: p < 0.01, F = 6.142; JunB: p < 0.05, F = 5.139) (Fig. 1G). Among the groups, the numbers of c-Fos or


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Fig. 2. Expression of c-Fos or JunB in CRH neurons of the PVN in S group (A and C) or W group (B and D). c-Fos (brown) and CRH (blue-gray) were detected by dual immunostaining (A and B), while JunB (brown) and CRH mRNA (purple) were detected by the combination of immunostaining and in situ hybridization (C and D). c-Fosexpressing CRH neurons (arrowheads) are abundant in the mpdd of the S group (A) but much fewer in the W group (B). JunB-expressing CRH neurons (arrowheads) are found in the mpdd of the S group (C) but in a reduced number in the W group (D). mpdd, dorsal body of the dorsal medial parvicellular part; pm, posterior magnocellular part. Scale bar = 50 ␮m.

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JunB-expressing nuclei were smaller (p < 0.05) in the W and P groups than in the S group. There is no significant difference between the B and S groups. The plasma CORT level did not differ among the groups (S group, 308.7 ± 25.0 ng/ml; W group, 251.7 ± 22.6 ng/ml; P group, 295.6 ± 18.8 ng/ml; and B group, 305.2 ± 22.1 ng/ml, n = 8 each).

3.4. c-Jun phosphorylation and AVP hnRNA expression after restraint, and the effects of white noise presentation pc-Jun-stained neurons existed in I, S and W groups (Fig. 4A–C). There was a significant difference in the mpdd (n = 4–5, p < 0.05;

3.3. c-Fos and JunB expression in CRH neurons and the effect of white noise presentation To detect c-Fos expression in CRH neurons, we analyzed the results by dual-immunostaining, because there was an unknown high-background found by the in situ hybridizaion step following c-Fos immunostaining. Many, but not all, CRH-stained neurons were shown to express c-Fos, especially in the mpdd of S group (Fig. 2A), whereas dual-stained neurons seemed fewer in W group (Fig. 2B). For JunB expression, we detected CRH by in situ hybridization. Dual-stained neurons were found in the mpdd of S group (Fig. 2C) and seemed fewer in W group (Fig. 2D). In semi-quantification, we first confirmed that the mean total number of histochemically-detected CRH neurons did not statistically differ between each PVN subdivision of S and W groups (data not shown). As shown in Fig. 3, c-Fos-expressing CRH neurons were significantly fewer (p < 0.05) only in the mpdd of the W group as opposed to the S group. c-Fos-expressing non-CRH neurons were also fewer in the mpdd and dp (p < 0.05, each), suggesting that there was activation of other parvicellular neurons. JunB-expressing CRH neurons were fewer (p < 0.05) only in the mpdd of the W group. On the other hand, JunB-expressing non-CRH neurons were fewer only in the mpdv (p < 0.05) of the W group, as compared to the S group.

Fig. 3. Expression of c-Fos or JunB in CRH neurons and non-CRH neurons in W and S groups. The significantly fewer c-Fos- or JunB-expressing CRH neurons in the W group (but not the S group) is found only in the mpdd. In non-CRH neurons, fewer c-Fos-expressing nuclei in the W group are seen in the mpdd and dp, while JunBexpressing nuclei are fewer in the mpdv in the W group. Data were expressed by mean ± SEM and analyzed for Welch t-test, *p < 0.05 vs. S group, n = 5 per group. dp, dorsal parvicellular part; mpdd, dorsal body of the dorsal medial parvicellular part; mpdv, ventral tail of the dorsal medial parvicellular part; pm, posterior magnocellular part.


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Fig. 4. pc-Jun immunostaining (brown) and AVP hnRNA in situ hybridization (purple) of the PVN of I (A and D), S (B and E), and W groups (C and F), as well as semiquantification (G). pc-Jun-stained nuclei are seen in parvicellular parts (A–C). AVP hnRNA signals are prominent in the mpdd of the S group (E) aside from the pm (D–F) of the three groups. Scale bar = 100 ␮m. Only in the mpdd, significantly more pc-Jun-stained nuclei exist in the S group (n = 5) than in the I (n = 4). However, there was no difference between the I and the W groups (n = 5). AVP hnRNA-expressing neurons in the mpdd and pm are more numerous in the S group (n = 9) than in the I (n = 9) and Q10 W (n = 10) groups. Data were expressed by mean ± SEM and analyzed for one-way ANOVA followed by Dunnett’s T3 post hoc test, **p < 0.01, *p < 0.05 vs. I group; †† p < 0.01, † p < 0.05 vs. S group. mpdd, dorsal body of the dorsal medial parvicellular part; mpdv, ventral tail of the dorsal medial parvicellular part; pm, posterior magnocellular part. (For interpretation of reference to color in this figure legend, the reader is referred to the web version of this article.) 350 351 352 353 354 355 356 357 358 359 360 361

F = 6.0), and more pc-Jun-stained nuclei were seen in the S group than in the I group (p < 0.05; Fig. 4G). In the W group, there was no significant difference, compared with S or I group. AVP hnRNA expression in the I group was found mainly in the pm of the I and W groups (Fig. 4D and F), but was also apparent in the mpdd (Fig. 4E). Among the three groups, there was significant difference only in the mpdd and the pm (the former, p < 0.05, F = 28.0; the latter, p < 0.05, F = 5.6). More AVP hnRNA-expressing nuclei were seen in the S group (p < 0.01 for the mpdd; p < 0.05 for the pm) than in the W and I groups, between which there was no difference (Fig. 4G).

3.5. Dual labeling detection of JunB with c-Fos or AVP hnRNA in the mpdd We explored spatial interrelationships between JunB and c-Fos, as well as between JunB and AVP hnRNA in the mpdd neurons of S group. Data show c-Fos-staining (Fig. 5A) or the AVP hnRNA signal (Fig. 5B) in JunB-stained nuclei. 4. Discussion Acute restraint is known to provoke c-Fos and AVP hnRNA expression in the CRH neurons correlated with the elevation of


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Fig. 5. Co-expressions of c-Fos (red) and JunB (green), as well as JunB (pink) and AVP hnRNA (purple) in the mpdd of S group. c-Fos staining (arrows in A) and JunB staining (arrows in B) co-localize (arrows in C) in DAPI-labeled nuclei of neurons (D). JunB is expressed in nuclei of mpdd neurons (E), some of which (arrows) also express AVP hnRNA (F). Scale bar = 50 ␮m for A–D, 100 ␮m for E and F. (For interpretation of reference to color in this figure legend, the reader is referred to the web version of this article.)

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plasma CORT level (Priou et al., 1993; Herman, 1995; Ma et al., 1997; Dayas et al., 1999; Chowdhury et al., 2000; Viau and Sawchenko, 2002; Girotti et al., 2006; Sterrenburg et al., 2012). As to these parameters, the present results agreed well with the previous studies, confirming the validity of our restraint paradigm for assessing the HPA response. Based upon this confirmation, we here report here an intriguing phenomenon in which simultaneous presentation of white or pink noise with restraint attenuates stress-induced c-Fos and JunB expression in the CRH neurons. Although a similar effect with other sensory modalities has already been reported (Ito et al., 2009; Martin and Timofeeva, 2010), the present study is the first to describe the modulatory effect of acoustic perception on the HPA response. The attenuation of c-Fos expression by noise presentation was found almost exclusively in the mpdd, suggesting its highly selective effect on neuroendocrine CRH neurons. Indeed, the present cell count data verified the decrease of c-Fos-expressing CRH neurons in the mpdd but not the mpdv, where neuroendocrine CRH neurons co-express methionine-enkephalin and sustain potent c-Fos expression even after repeated immobilization (Dumont et al., 2000; Viau and Sawchenko, 2002). Thus, the present study suggests that the perception of white noise can affect certain

c-Fos-mediated events, particularly neuroendocrine CRH neurons. Unlike other sensory stimuli (green odor: Ito et al., 2009; sucrose: Martin and Timofeeva, 2010), we observed no signs of change in the plasma CORT level. This suggests that although the mpdd CRH neurons can respond to restraint stress in two mutually independent ways: (1) CRH release and transcription, and (2) c-Fos and JunB expression, noise presentation influences exclusively expression of the IEGs. Therefore, the noise-related attenuation of c-Fos response seems to be unrelated to stress-evoked CRH and ACTH release by the CRH neurons. In regard to this, intra-PVN infusion studies show that c-fos mRNA and c-Fos expression is evoked by restraint even after infusing dexamethasone or bovine serum albumin-conjugated CORT, both of which block restraintinduced CRH hnRNA expression and an increase in the plasma ACTH level (Evanson et al., 2010; Weiser et al., 2011). This sustainable c-Fos expression may reflect the disinhibition of GABAergic tone (Herman and Cullinan, 1997; Bali and Kovács, 2003) rather than the blocking of negative feedback regulation. The attenuation of c-Fos expression caused by noise presentation might be responsible for the GABAergic negative tone on the mpdd CRH neurons. Thus, it may be that acoustic stimuli attenuate CRH neuron activation by a diverse cellular mechanism from the processing of


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olfactory or gustatory information. Regarding the neuronal mechanism, however, there is no information about the neural pathway through which green odor affects stress-related CORT release (Ito et al., 2009). In contrast, sucrose ingestion has been reported to activate the mesolimbic dopaminergic system (Hajnal et al., 2004; Norgren et al., 2006), with the enhanced dopamine discharge in the nucleus accumbens shell. Therefore, sucrose as a chemosensory award might counteract with CRH secretion triggered by restraint, although the underlying neural pathway still remains unclear. As to the acoustic stimuli, a recent human study reported that appreciation of music as an abstract stimulus evokes dopamine release in the nucleus accumbens (Salimpoor et al., 2011). However, it is unknown whether or not noise presentation acts as a reward stimulus for rats. To date, JunB has attracted less interest than c-Fos in terms of HPA response, despite its concomitant transient expression with c-Fos in the PVN by acute restraint (Stamp and Herbert, 1999). As for JunB, the present findings were as follows: (1) expression in the mpdd CRH neurons; (2) attenuated expression by white noise presentation; and (3) co-expression with c-Fos or AVP hnRNA in mpdd neurons. An in vitro study using a neuroblastoma cell line, which expresses endogenous AVP mRNA, showed that the c-Fos:JunB heterodimer most potently induces avp transcription among various Fos/Jun family protein dimers as activator protein 1 (AP1) (Yoshida et al., 2006). Altogether, the present results suggest that in our restraint paradigm, JunB and c-Fos are candidates for the AP1 partner involved in IEG-mediated events such as avp transcription in the mpdd CRH neurons. As for c-Jun in the PVN, previous studies have reported its persistent expression (Herdegen et al., 1995), without change in the c-Jun-expressing neuron number after acute restraint (Stamp and Herbert, 1999). An in situ hybridization study described only a small elevation of c-jun mRNA expression in the entire PVN (Imaki et al., 1996). Thus, no studies addressed the stress-related activation of c-Jun protein at the cellular level. In this respect, by employing a pc-Jun antibody against peptide having phosphorylated Ser 63 distinguishable from JunD (Dunn et al., 2002), we showed that pc-Jun positive neurons increase only in the mpdd by restraint, but were unchanged in any PVN subdivisions by white noise presentation. This is the first report to verify the stressinduced phosphorylation of c-Jun in PVN neurons, suggesting that pc-Jun is not another AP1 partner of c-Fos or JunB that is responsible for the attenuated expression of AVP hnRNA. Regarding the time course of stress response, previous studies have reported that c-Fos expression is maximal at 1–2 h after a 5-min ether inhalation (in accordance with the timing of peak AVP hnRNA), but not the CRH hnRNA level, as revealed by both population densitometry and neuron counts (Kovács and Sawchenko, 1996). A study dealing with restraint stress reported that CRH and AVP hnRNA levels significantly increased at 1 and 2 h, respectively, after the onset of restraint (Ma et al., 1997). In this regard, the present semi-quantification showed that AVP hnRNA-expressing neurons in the mpdd 2 h after the onset of restraint are fewer in the rats presented with white noise, as compared to those without white noise. Also at this time, c-Fos or JunB-expressing CRH neurons were fewer only in the mpdd with white noise presentation. Taken with co-expression of c-Fos and JunB, as well as the JunB and AVP hnRNA in mpdd neurons of restrained rats, the attenuation of AVP hnRNA expression may result from the reduction in AP1 activity, attributable to the attenuated expression of both c-Fos and JunB in the mpdd CRH neurons. A similar change in the number of AVP hnRNA-expressing neurons was found in the pm, suggesting a putative effect on hormone production in magnocellular AVP neurons. As for the neuronal organization of the PVN, some parvicellular neurons are known to exist ectopically in the pm (Kiss et al., 1991), intermingling with magnocellular neurons at the border of the mpdd and the pm. Therefore, the possibility that we also

counted such ectopic parvicellular neurons cannot be excluded. Recent evidence suggests that magnocellular AVP neurons are also activated by psychological stressors such as social defeat, leading to the central release of AVP displaying anxiety-related behavior (Wotjak et al., 1996; Engelman et al., 2004). Therefore, the question of whether or not the data of the pm are relevant remains to be elucidated, because only a small c-Fos or JunB expression was found in this subdivision of our paradigm. Besides CRH neurons, white noise presentation attenuated c-Fos expression in the mpdd and dp, and JunB expression in the mpdv of non-CRH neurons, as compared with S group. In light of the previous studies, at least part of these non-CRH neurons may correspond to hypophysiotropic or autonomic peptidergic neurons (Dumont et al., 2000; Viau and Sawchenko, 2002; Simmons and Swanson, 2009). However, we have no appropriate interpretation for the present results, because little is known to stress-related functional roles of the peptidergic neurons in these subdivisions of the PVN. A primary role of AVP in the HPA axis is to potentiate synergistically the activity of CRH to release pituitary ACTH (Gillies et al., 1982) via an AVP receptor (V1b) (Tanoue et al., 2004), to exert the full response to stressors. Previous studies have reported an increase, both in the production and release of AVP in the mpd CRH neurons by repeated restraint (Whitnall, 1993; Aguilera, 1994; Herman, 1995) accompanied by the V1b receptor up-regulation (Tizabi and Aquilera, 1992; Rabadan-Diehl et al., 1995; Aguilera and Rabadan-Diehl, 2000). This indicates that AVP rather than CRH acts as a dominant ACTH secretagogue under chronic stress conditions. In light of these studies, one possible interpretation of the present results is that an acoustic stimulus such as white noise has the potency to attenuate the restraint-evoked AVP hnRNA expression as an adaptive response to the prospective homotypic stressor. The study of noise has, to date, revealed some effects on the brain functionality of rats (Lai, 1987; Arankowsky-Sandoval et al., 1992; Campeau and Watson, 1997; Campeau et al., 2002; Kraus and Canlon, 2012). As for the HPA axis, the effect of white noise is reported to be intensity-dependent; a high intensity (90 and 105 dB of sound pressure level) of noise causes c-fos mRNA expression in the PVN and elevates the plasma CORT level as an acoustic stressor, while moderate intensity (70 and 80 dB) noise has no stressor effect but prolongs sleep duration (Campeau and Watson, 1997). Moreover, a tone of 2-kHz noise at 75–80 dB is suggested to facilitate REM sleep (Arankowsky-Sandoval et al., 1992). The present results showed that restraint-induced expression of IEGs and AVP hnRNA is attenuated by presentation of white and pink noise but not blue noise, suggesting an effect specific for low-frequency components of noise. Therefore, it is likely that the white noise (70 dB, 20 Hz–20 kHz) used in the present study also induced sleep in the rats even during restraint. A key aspect of stress is a heightened level of arousal (Berridge et al., 2010), to which lateral hypothalamic orexinergic neurons contribute as part of wake-promoting systems (Datta and MacLean, 2007; Berridge et al., 2010; Inutsuka and Yamanaka, 2013). Intracerebroventricular injection of this peptide evokes c-Fos expression in parvicellular neurons of the PVN in rats (Date et al., 1999), with elevation in the plasma levels of ACTH and CORT (Jászberényi et al., 2000; Kuru et al., 2000). Therefore, another explanation for the present results might be that the attenuation of IEGs and AVP hnRNA expression is partly due to the decreased orexinergic stimulatory tone to the mpdd CRH neurons under the inhibition of GABAergic neurons in the ventrolateral preoptic nucleus (Sakurai et al., 2005). 5. Conclusion We investigated the effect of acoustic stimulus presentation on the HPA response to acute restraint stress in rats. Presentation of


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white noise during restraint does not change plasma CORT level, but attenuates c-Fos and JunB expression and the resultant avp gene transcription in neuroendocrine CRH neurons in the mpdd of the PVN. These results suggest that restraint-induced AP1 formation is influenced by concurrent acoustic perception, resulting in the reduction in AVP production as a chronic stress response.

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This study was supported in part by a Grant-in-Aid (no. Q9 21240020) for Scientific Research (A), from the Ministry of Edu553 cation, Culture Sports, Science, and Technology of Japan (S.H.). 554 552

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Chronic unpredictable stress and long-term ovariectomy affect arginine-vasopressin expression in the paraventricular nucleus of adult female mice.

Angiotensinergic neurotransmission in the paraventricular nucleus of the hypothalamus modulates the pressor response to acute restraint stress in rats.

Stimulation of the hypothalamic paraventricular nucleus produces analgesia not mediated by vasopressin or endogenous opioids.

Restraint stress attenuates nicotine's locomotor stimulant but not discriminative stimulus effects in rats.

Glutamate microinjection into the hypothalamic paraventricular nucleus attenuates ulcerative colitis in rats.

Gastrin-17 injected into the hypothalamic paraventricular nucleus can induce gastric acid secretion in rats.

Gestational hypoxia modulates expression of corticotropin-releasing hormone and arginine vasopressin in the paraventricular nucleus in the ovine fetus.

Regulation of basal corticotropin-releasing hormone and arginine vasopressin messenger ribonucleic acid expression in the paraventricular nucleus: effects of selective hypothalamic deafferentations.

Chronic infusion of lisinopril into hypothalamic paraventricular nucleus modulates cytokines and attenuates oxidative stress in rostral ventrolateral medulla in hypertension.

Noradrenergic activation in the paraventricular nucleus during acute and chronic immobilization stress in rats: an in vivo microdialysis study.

Neurons in the hypothalamic paraventricular nucleus mediate the serotonergic stimulation of renin secretion.

Chronic stress enhances vasopressin but not corticotropin-releasing factor secretion during hypoglycemia.

Exercise training attenuates hypertension and cardiac hypertrophy by modulating neurotransmitters and cytokines in hypothalamic paraventricular nucleus.

Food entrains clock genes but not metabolic genes in the liver of suprachiasmatic nucleus lesioned rats.

The magnocellular arginine-vasopressin mRNA responds differently to food deprivation between the supraoptic and paraventricular nuclei of the hypothalamus in adrenalectomized rats with low corticosterone replacement.

Expression of immediate-early genes in the dorsal cochlear nucleus in salicylate-induced tinnitus.

Repeated stress enhances vasopressin synthesis in corticotropin releasing factor neurons in the paraventricular nucleus.

Gastric motility and food intake in rats after lesions of hypothalamic paraventricular nucleus.

Stimulation of adrenocorticotropin secretion by insulin-induced hypoglycemia in the developing rat involves arginine vasopressin but not corticotropin-releasing factor.

Vasopressin, corticosterone levels, and gastric ulcers during food-restriction stress.

Arginine Vasopressin Injected into the Dorsal Motor Nucleus of the Vagus Inhibits Gastric Motility in Rats.

Hypothyroidism increases vasoactive intestinal polypeptide (VIP) immunoreactivity and gene expression in the rat hypothalamic paraventricular nucleus.

Adrenergic projections from the lower brainstem to the hypothalamic paraventricular nucleus, the lateral hypothalamic area and the central nucleus of the amygdala in rats.

Intermedin in the paraventricular nucleus attenuates cardiac sympathetic afferent reflex in chronic heart failure rats.