G Model
ARTICLE IN PRESS
NSL 31354 1–6
Neuroscience Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
1
Activation of hypothalamic oxytocin neurons following tactile stimuli in rats
2
3
4
Q2
Shota Okabe, Masahide Yoshida, Yuki Takayanagi, Tatsushi Onaka ∗ Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University, Shimotsuke-shi, Tochigi-ken 329-0498, Japan
5 6
h i g h l i g h t s
7 8
• Stroking stimuli activated hypothalamic paraventricular oxytocin neurons. • Rats emitted 50-kHz ultrasonic vocalizations during stroking stimuli. • Pleasant stroking is thought to activate hypothalamic oxytocin neurons.
9 10 11 12
a r t i c l e
13 28
i n f o
a b s t r a c t
14
Article history: Received 21 April 2015 Received in revised form 16 May 2015 Accepted 25 May 2015 Available online xxx
15 16 17 18 19 20
27
Keywords: Oxytocin Stroking Touch Fos protein Ultrasonic vocalizations Hypothalamus
29
1. Introduction
21 22 23 24 25 26
Gentle touching or stroking has anxiolytic actions and contributes to the establishment of an intimate relationship between individuals. Oxytocin administration also has anxiolytic actions and facilitates social behaviors. In this study, we examined effects of stroking stimuli on activation of oxytocin neurons and emission of 50-kHz ultrasonic vocalizations, an index of positive emotion, in rats. The number of oxytocin neurons expressing Fos protein was increased in the hypothalamus, especially in the dorsal zone of the medial parvicellular part of the paraventricular nucleus. The number of 50-kHz ultrasonic vocalizations was also increased. These findings suggest that pleasant sensory stimuli activate hypothalamic oxytocin neurons. © 2015 Published by Elsevier Ireland Ltd.
Gentle touching or stroking induces a pleasant sensation via activation of slow-conducting unmyelinated nerves [1,2]. Affective touch plays an important role in affiliative behaviors including parental care and pair-bonding to reduce anxiety [3], to induce analgesia [4] and to contribute to the establishment of an intimate relationship [5]. Information of this affective-motivational dimension of touch is processed in social brain regions including the medial prefrontal cortex, cingulate cortex, insula, and superior temporal sulcus [6]. However, subcortical regions that are activated by affective touch are largely unclear. Rats emit ultrasonic vocalizations dependent on the their emotional states. In aversive situations such as during predator exposure, lower frequency vocalizations, 22-kHz vocalizations, are emitted [7]. In appetitive situations such as during social investi-
30 31 32 33 34 35 36 37 38 39 40 41 42 43
Q3
∗ Corresponding author. Tel.: +81 285587318; fax: +81 285448147. E-mail address: [email protected] (T. Onaka).
gation and rough-and-tumble play, higher frequency vocalizations, 50-kHz vocalizations, are elicited [7]. Thus, 22-kHz and 50-kHz vocalizations have been used as indices of negative and positive states of rats, respectively [7]. Oxytocin, which is mainly synthesized in the hypothalamus and the bed nucleus stria terminalis (BNST), has been shown to be associated with social behaviors [8–15], to induce anti-stress or anxiolytic actions [16] and to induce analgesia [17]. Oxytocin is released from the neurohypophysis into the blood at birth and during lactation and acts on peripheral tissues. Oxytocin has also been shown to be released from dendrites or cell bodies as well as from axonal terminals of oxytocin neurons within the brain following appropriate stimuli [18]. Massage or non-noxious tactile stimulation induces an increase in plasma oxytocin [19–21]. However, the localization of oxytocin neurons activated by non-noxious sensory stimuli has remained to be identified. In this study, we examined the emission of ultrasonic vocalizations, an index of emotional states, and expression of Fos protein, an index of neuronal activation, in oxytocin neurons of the hypothalamus and BNST following tactile stimuli in rats.
http://dx.doi.org/10.1016/j.neulet.2015.05.055 0304-3940/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
G Model NSL 31354 1–6
ARTICLE IN PRESS S. Okabe et al. / Neuroscience Letters xxx (2015) xxx–xxx
2 64
2. Methods
65
2.1. Animals
76
Twenty-four male rats (9 weeks old, Iar: Long–Evans, Institute for Animal Reproduction, Ibaraki, Japan) were housed individually under a 12:12 h light/dark cycle (lights on at 7:30 am) at 22 ± 2 ◦ C and 55 ± 15% relative humidity. Food and water were available ad libitum. Animal experiments were carried out after receiving approval from the Animal Experiment Committee of Jichi Medical University and were in accordance with the Institutional Regulations for Animal Experiments and Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology.
77
2.2. Massage-like stroking
66 67 68 69 70 71 72 73 74 75
87
Rats were placed on the experimenter’s lap and given stroking stimuli on the dorsal part of their bodies by the experimenter’s hand with a cotton glove at a speed of 8.76 ± 0.17 cm/s (mean ± SEM, n = 8) and a frequency of 0.62 ± 0.01 Hz (mean ± SEM, n = 8) for 5 min without immobilization of their bodies. The speed and frequency of the stroking stimuli were monitored by use of a video camera. The force applied by the manual-stroking stimuli was relatively mild and was estimated to be less than 250 mN/cm2 . Cotton gloves were used for stroking to reduce friction and to make stroking as smooth as possible.
88
2.3. Recording and analysis of ultrasonic vocalizations
78 79 80 81 82 83 84 85 86
89 90 91 92 93 94 95 96 97 98 99 100 101 102
103 104
105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121
Ultrasonic vocalizations recording was performed by use of a 1/4-inch measurement-grade microphone (Type 4158N, Aco, Tokyo, Japan) designed for measurements of sound pressure levels between 20 Hz and 100,000 Hz. The microphone was placed at a distance of approximately 30 cm from the rats and was connected to a sensor amplifier (SR-2200, Ono Sokki, Kanagawa, Japan) and an AD–DA converter (USB-6251, NI, Texas, USA). Acoustic data were recorded at a sampling rate of 200 kHz and with 16 bits by an Avisoft RECORDER (Version 4.2, Avisoft Bioacoustics, Berlin, Germany) and analyzed by Avisoft SASLab Pro (Version 5.2, Avisoft Bioacoustics). Spectrograms were generated with a fast Fourier transform length of 256 points and an overlap of 50%. The numbers of 50kHz and 22-kHz vocalizations were counted manually according to previous reports [22,23]. 2.4. Immunohistochemical detection of fos protein in oxytocin-immunoreactive (-ir) neurons Ninety minutes after termination of stroking or holding stimuli, the rats were anesthetized with Avertin (tribromoethanol; 200 mg/kg, i.p.) and perfused transcardially with heparinized saline (2 U/mL) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min. The brains were immediately removed from the skulls, post-fixed in 4% paraformaldehyde overnight, placed in 30% sucrose in 0.1 M phosphate buffer until they sank, and frozen in dry ice. The hypothalamic part of each frozen brain was sectioned coronally at 30 m and processed for immunochemical detection of Fos protein and oxytocin as described previously with modifications [24]. In brief, sections were incubated with a rabbit polyclonal antibody raised against the Fos peptide sequence (Ab-5; Oncogene Science; Cambridge, MA, USA) diluted at 1:10,000 for 2 days at 4 ◦ C. Immunoreactivity was visualized by sequential overnight incubation with peroxidase-labeled goat anti-rabbit IgG (diluted at 1:500; Vector Laboratories, Inc., Burlingame, CA, USA) at 4 ◦ C and 3,3-diaminobenzidine tetrahydrochloride (DAB)
Fig. 1. Ultrasonic vocalizations during stroking and holding stimuli. A spectrogram of 50-kHz vocalizations in stroking group (A) and the numbers of 50-kHz and 22-kHz vocalizations during stroking (B) and holding (C) are shown. Rats emitted 50-kHz ultrasonic vocalizations more often than 22-kHz vocalizations during stroking and holding stimuli. *, P < 0.05. n = 8 per group.
with nickel sulphate. The sections were then processed for detection of oxytocin using an antibody against oxytocin (diluted at 1:1,000,000; Peninsula Laboratories International, San Carlos, CA, USA). After two days of incubation at 4 ◦ C with the first antibody, sections were incubated with biotinylated anti-guinea pig IgG (diluted at 1:750; Vector Laboratories) for 2 h at room temperature and with avidin biotinylated horseradish peroxidase complex (diluted at 1:50; Vector Laboratories) for 30 min. Oxytocin was visualized with DAB. Sections containing hypothalamic paraventricular nuclei (PVN) (14 sections), supraoptic nuclei (SON) (15 sections) or BNST (8 sections) were examined at intervals of 120 m in each rat. Areas of the brain were identified according to brain atlases [25,26] 2.5. Groups and experimental design All rats received 5-min stroking stimuli once a day for 7 days. On the following day, rats were assigned to one of three groups: stroking group (n = 8), holding group (n = 8), and non-touch group (n = 8). In the stroking group, rats were placed on the experimenter’s lap and given massage-like stroking for 5 min. In the holding group, rats were handled in the same manner as rats in the stroking group for 5 min without receiving stroking stimuli. In the non-touch group, rats were kept in their individual home cages. In the stroking and holding groups, ultrasonic vocalizations were monitored by use of an ultrasound microphone. After 5-min manipulation, rats were returned to their home cages. Ninety minutes after termination of stroking or holding stimuli, the rats were perfused with 4% paraformaldehyde following deep anesthesia. 2.6. Statistical analysis Data are expressed as means ± SEM. The numbers of 50-kHz and 22-kHz ultrasonic vocalizations were analyzed by Wilcoxon’s signed rank test or the Mann–Whitney U test. The number of immunoreactive cells was analyzed by one-way ANOVA followed
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
122 123 124 125 126 127 128 129 130 131 132 133 134
135
136 137 138 139 140 141 142 143 144 145 146 147 148 149
150
151 152 153 154
G Model NSL 31354 1–6
ARTICLE IN PRESS S. Okabe et al. / Neuroscience Letters xxx (2015) xxx–xxx
3
Fig. 2. Expression of Fos protein in oxytocin-immunoreactive (-ir) neurons following stroking stimuli. Oxytocin-ir neurons expressing Fos protein in the bed nucleus of the stria terminalis (BNST) (A1, B1, C1), the supraoptic nucleus (SON) (A2, B2, C2), and the paraventricular nucleus (PVN) (A3, B3, C3) in the stroking (A1–A3), holding (B1-B3), and non-touch groups (C1–C3) and the percentages of oxytocin-ir neurons expressing Fos protein in the BNST (D), SON (E), and PVN (F) are shown. The percentages of oxytocin-ir neurons expressing Fos protein were significantly increased in the SON and PVN after stroking as compared with the non-touch group. Brown cytoplasmic reactions indicate oxytocin immunoreactivity, and dark nuclear reactions indicate Fos immunoreactivity. Areas enclosed by black rectangles in A2 and A3 are shown at a higher magnification. The arrowheads indicate double-labeled neurons. Scale bar in A1 = 50 m. Scale bar in black rectangles = 12.5 m. *, P < 0.05. n = 8 per group.
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
G Model NSL 31354 1–6
ARTICLE IN PRESS S. Okabe et al. / Neuroscience Letters xxx (2015) xxx–xxx
4
Fig. 3. Expression of Fos protein in oxytocin-ir neurons in the dorsal zone of the medial parvicellular part of the PVN following stroking stimuli. Oxytocin-ir neurons expressing Fos protein in the dorsal zone of the medial parvicellular part of the PVN in the stroking (A), holding (B), and non-touch groups (C), and the percentages of oxytocin-ir neurons expressing Fos protein in the dorsal zone of the medial parvicellular part of the PVN (D) are shown. The percentage of oxytocin-ir cells expressing Fos protein was significantly increased after stroking as compared with non-touch or holding group. The arrowhead indicates a double-labeled neuron. Scale bar = 5 m. *, P < 0.05. n = 8 per group.
156
by the Tukey–Kramer post hoc test. P < 0.05 was considered statistically significant.
more than that of 22-kHz vocalizations in both groups (Fig. 1B and C; P < 0.05, Wilcoxon’s signed rank test).
157
3. Results
3.2. Expression of Fos protein in oxytocin-immunoreactive (-ir) neurons
158
3.1. Ultrasonic vocalizations
155
159 160 161
In the stroking group, rats emitted 50-kHz vocalizations (Fig. 1A). In addition, in the holding group, rats emitted 50-kHz vocalizations. The number of 50-kHz vocalizations was significantly
In the BNST, SON and PVN, there were no significant differences in the total number of oxytocin-ir cells or Fos protein-ir cells among the stroking, holding and non-touch control groups (data not shown). However, compared to those in the non-touch control group, the percentages of oxytocin-ir cells expressing Fos protein
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
162 163
164 165
166 167 168 169 170
G Model NSL 31354 1–6
ARTICLE IN PRESS S. Okabe et al. / Neuroscience Letters xxx (2015) xxx–xxx
171 172 173 174 175 176 177 178 179
180
181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233
in the stroking group were significantly higher in the SON (Fig. 2A2, B2, C2, and E; P < 0.05) and PVN (Fig. 2A3, B3, C3, and F; P < 0.05) but not in the BNST (Fig. 2A1, B1, C1, and D). Oxytocin-ir cells expressing Fos protein were observed in the PVN, especially in the dorsal zone of the medial parvicellular part. Compared to those in the non-touch control and holding group, the percentages oxytocin-ir cells expressing Fos protein in the stroking group were significantly higher in the dorsal zone of the medial parvicellular part (Fig. 3; P < 0.05).
4. Discussion Tactile stimuli or massage have been shown to increase plasma oxytocin concentrations in humans [27]. In the present study, we demonstrated that gentle stroking activated oxytocin neurons in PVN and SON neurons of rats. During stroking stimuli, the animals emitted 50-kHz ultrasonic vocalizations, which have been associated with positive states [7]. The findings suggest that affective sensory stimuli activate hypothalamic oxytocin neurons. The present study showed that gentle stroking induced expression of Fos protein, an index of neuronal activation, in oxytocin neurons of the hypothalamic PVN and SON. Plasma oxytocin concentrations were not measured in the present study. However, following one-minute stroking stimuli applied to the whole bodies of rats held by the experimenter’s hand, plasma oxytocin concentrations were slightly but significantly increased (Wistar strain, 11.6 ± 0.9 pg/ml for the control group, 14.5 ± 0.8 pg/ml for the stroking group, n = 6, P < 0.05, t-test, unpublished data). Although the procedures used for stroking stimuli were slightly different from the present study (held by hand vs free-moving, applied to the whole body vs the back, Wistar strain vs Long–Evans strain), it is likely that oxytocin release into plasma might also have been facilitated in response to non-noxious tactile stimuli in the present study. In the present study, oxytocin neurons activated following tactile stimuli were mainly located in the dorsal zone of the medial parvicellular part of the PVN (Fig. 3). Oxytocin neurons in the PVN have been shown to project to the ventral tegmental areas, periaqueductal gray, nucleus tractus solitarius, and spinal cord [28,29,30]. These brain areas are associated with processing for reward [31], pain [32], and cardiovascular control [33]. Both oxytocin application and non-noxious sensory stimuli modulate positive emotional behavior [3,21], induce analgesia [17,20], facilitate affiliative behavior [5,34], and change cardiovascular parameters [35,36]. It is thus interesting to speculate that oxytocin is involved in these actions of non-noxious sensory stimulation by acting on the ventral tegmental areas, periaqueductal gray, nucleus tractus solitarius, or spinal cord. Further research is necessary to clarify the physiological roles of oxytocin in effects of tactile stimuli. In the present study, rats emitted 50-kHz vocalizations while being held without receiving stroking as well as during stroking stimuli. Emission of 50 kHz vocalizations is considered to reflect positive emotion [7]. The animals in the present study received stroking stimuli while being held for 7 days, and 50-kHz ultrasonic vocalizations have been shown to be emitted not only during affiliative social contact but also during anticipation of social contact [37] or reward anticipation [38]. It is thus possible that holding stimuli might have induced anticipation of stroking stimuli and induced 50-kHz ultrasonic vocalizations in the present study. Oxytocin neurons are activated by various kinds of stressful stimuli [13]. The data obtained in this study showing that stroking stimuli activated oxytocin neurons in the PVN might be explained by stress due to stroking stimuli. However, it is not likely. In the present study, animals emitted 50-kHz ultrasonic vocalizations during stroking. On the other hand, the number of
5
22-kHz ultrasonic vocalizations, which reflect negative emotions [7], was significantly less than that of 50-kHz ultrasonic vocalizations during stroking stimuli. Consistent with the idea that stroking stimuli were not stressful in the present study, the number of Fos-positive paraventricular non-oxytocin neurons, which are thought to include CRF-synthesizing neurons, was not significantly increased following stroking stimuli (data not shown). Non-noxious sensory stimuli have been shown to increase plasma or urinary oxytocin concentrations in rats [21], dogs [39], and humans [27]. Massage-like hand movement of a baby induces an increase in plasma oxytocin concentration in the mother [40]. From all these data, we conclude that affiliative tactile stimuli activate oxytocin neurons in the hypothalamus. Acknowledgments This work was supported in part by grants from the Japan Soci- Q4 ety for the Promotion of Science (15K18978 to S.O.,26460322 to Y.T., 26460321 to M.Y. and 25118008, 26293049 and 15K15042 to T.O.,) and by the the MEXT (the Ministry of Education, Culture, Sports, Science and Technology of Japan -Supported Program for the Strategic Research Foundation at Private Universities, 2011-2015 (Cooperative Basic and Clinical Research on Circadian Medicine) References [1] H. Olausson, Y. Lamarre, H. Backlund, C. Morin, B.G. Wallin, G. Starck, S. Ekholm, I. Strigo, K. Worsley, Å.B. Vallbo, M.C. Bushnell, Unmyelinated tactile afferents signal touch and project to insular cortex, Nat. Neurosci. 5 (2002) 900–904. [2] L.S. Löken, J. Wessberg, I. Morrison, F. McGlone, H. Olausson, Coding of pleasant touch by unmyelinated afferents in humans, Nat. Neurosci. 12 (2009) 547–548. [3] A. Gallace, C. Spence, The science of interpersonal touch: an overview, Neurosci. Biobehav. Rev. 34 (2010) 246–259.
234 235 236 237 238 239 240 241 242 243 244 245 246
247
248 249 250 251 252 253 254
255
256 257 258 259 260 261 262 263 264 265
[4] G. Agren, T. Lundeberg, K. Uvnäs-Moberg, A. Sato, The oxytocin antagonist 1-deamino-2-D-tyr-(oet)-4-thr-8-orn-oxytocin reverses the increase in the withdrawal response latency to thermal, but not mechanical nociceptive stimuli following oxytocin administration or massage-like stroking in rats, Neurosci. Lett. 187 (1995) 49–52. [5] R.I.M. Dunbar, The social role of touch in humans and primates: behavioural function and neurobiological mechanisms, Neurosci. Biobehav. Rev. 34 (2010) 260–268. [6] I. Gordon, A.C. Voos, R.H. Bennett, D.Z. Bolling, K.A. Pelphrey, M.D. Kaiser, Brain mechanisms for processing affective touch, Hum. Brain Mapp. 34 (2013) 914–922. [7] S.M. Brudzynski, Ethotransmission: communication of emotional states through ultrasonic vocalization in rats, Curr. Opin. Neurobiol. 23 (2013) 310–317. [8] Z.R. Donaldson, L.J. Young, Oxytocin, vasopressin, and the neurogenetics of sociality, Science 7 (2008) 900–904. [9] A.M. Kelly, J.L. Goodson, Social functions of individual vasopressin-oxytocin cell groups in vertebrates: what do we really know? Front. Neuroendocrinol. 35 (2014) 512–529. [10] R. Stoop, Neuromodulation by oxytocin and vasopressin in the central nervous system as a basis for their rapid behavioral effects, Curr. Opin. Neurobiol. 29 (2014) 187–193. [11] C.S. Carter, Oxytocin pathways and the evolution of human behavior, Annu. Rev. Psychol. 65 (2014) 17–39. [12] H.S. Knobloch, V. Grinevich, Evolution of oxytocin pathways in the brain of vertebrates, Front. Behav. Neurosci. 8 (2014) 31. [13] T. Onaka, Y. Takayanagi, M. Yoshida, Roles of oxytocin neurones in the control of stress energy metabolism, and social behaviour, J. Neuroendocrinol. 24 (2012) 587–598. [14] H. Higashida, S. Yokoyama, M. Kikuchi, T. Munesue, CD38 and its role in oxytocin secretion and social behavior, Horm. Behav. 61 (2012) 351–358. [15] M. Nagasawa, S. Mitsui, S. En, N. Ohtani, M. Ohta, Y. Sakuma, T. Onaka, K. Mogi, T. Kikusui, Oxytocin-gaze positive loop and the coevolution of human-dog bonds, Science 348 (2015) 333–336. [16] M. Yoshida, Y. Takayanagi, K. Inoue, T. Kimura, L.J. Young, T. Onaka, K. Nishimori, Evidence that oxytocin exerts anxiolytic effects via oxytocin receptor expressed in serotonergic neurons in mice, J. Neurosci. 29 (2009) 2259–2271. [17] M. Petersson, P. Alster, T. Lundeberg, K. Uvnäs-Moberg, Oxytocin increases nociceptive thresholds in a long-term perspective in female and male rats, Neurosci. Lett. 212 (1996) 87–90.
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306
G Model NSL 31354 1–6 6 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340
ARTICLE IN PRESS S. Okabe et al. / Neuroscience Letters xxx (2015) xxx–xxx
[18] G. Leng, M. Ludwig, Neurotransmitters and peptides: whispered secrets and public announcements, J. Physiol. 586 (2008) 5625–5632. [19] S. Stock, K. Uvnäs-Moberg, Increased plasma levels of oxytocin in response to afferent electrical stimulation of the sciatic and vagal nerves and in response to touch and pinch in anaesthetized rats, Acta. Physiol. Scand. 132 (1988) 29–34. [20] K. Uvnäs-Moberg, G. Bruzelius, P. Alster, T. Lundeberg, The antinociceptive effect of non-noxious sensory stimulation is mediated partly through oxytocinergic mechanisms, Acta. Physiol. Scand. 149 (1993) 199–204. [21] K. Uvnäs-Moberg, L. Handlin, M. Petersson, Self-soothing behaviors with particular reference to oxytocin release induced by non-noxious sensory stimulation, Front. Psychol. 5 (2015) 1529. [22] S.M. Brudzynski, Pharmacological and behavioral characteristics of 22khz alarm calls in rats, Neurosci. Biobehav. Rev. 25 (2001) 611–617. [23] J. Burgdorf, R.A. Kroes, J.R. Moskal, J.G. Pfaus, S.M. Brudzynski, J. Panksepp, Ultrasonic vocalizations of rats (Rattus norvegicus) during mating, play, and aggression: behavioral concomitants, relationship to reward, and self-administration of playback, J. Comp. Psychol. 122 (2008) 357–367. [24] L. Zhu, T. Onaka, Involvement of medullary A2 noradrenergic neurons in the activation of oxytocin neurons after conditioned fear stimuli, Eur. J. Neurosci. 16 (2002) 2186–2198. [25] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, sixth ed., Academic Press, San Diego, 2007. [26] L.W. Swanson, Brain Maps: Structure Of The Rat Brain, second ed., Elsevier, Amsterdam, 1999. [27] V. Morhenn, L.E. Beavin, P.J. Zak, Massage increases oxytocin and reduces adrenocorticotropin hormone in humans, Altern. Ther. Health Med. 18 (2012) 11–18. [28] M.V. Sofroniew, Projections from vasopressin oxytocin, and neurophysin neurons to neural targets in the rat and human, J. Histochem. Cytochem. 28 (1980) 475–478. [29] T.A.P. Roeling, J.G. Veening, J.P.W. Peters, M.E.J. Vermelis, R. Nieuwenhuys, Efferent connections of the hypothalamic grooming area in the rat, Neuroscience 56 (1993) 199–225.
[30] J.D. Breton, P. Veinante, S. Uhl-Bronner, A.M. Vergnano, M.J. Freund-Mercier, R. Schlichter, P. Poisbeau, Oxytocin-induced antinociception in the spinal cord is mediated by a subpopulation of glutamatergic neurons in lamina I-II which amplify GABAergic inhibition, Mol. Pain 4 (2008). [31] S. Lammel, B.K. Lim, R.C. Malenka, Reward and aversion in a heterogeneous midbrain dopamine system, Neuropharmacology 76 (2014) 351–359. [32] M.M. Heinricher, I. Tavares, J.L. Leith, B.M. Lumb, Descending control of nociception: specificity, recruitment and plasticity, Brain Res. Rev. 60 (2009) 214–225. [33] A. Krassioukov, V.E. Claydon, The clinical problems in cardiovascular control following spinal cord injury: an overview, Prog. Brain Res. 152 (2005) 223–229. [34] T.R. Insel, The challenge of translation in social neuroscience: a review of oxytocin, vasopressin, and affiliative behavior, Neuron 65 (2010) 768–779. [35] M. Petersson, P. Alster, T. Lundeberg, K. Uvnäs-Moberg, Oxytocin causes a long-term decrease of blood pressure in female and male rats, Physiol. Behav. 60 (1996) 1311–1315. [36] M. Kurosawa, T. Lundeberg, G. Ågren, I. Lund, K. Uvnäs-Moberg, Massage-like stroking of the abdomen lowers blood pressure in anesthetized rats: influence of oxytocin, J. Auton. Nerv. Syst. 56 (1995) 26–30. [37] A.R. Willey, L.P. Spear, Development of anticipatory 50 kHz usv production to a social stimuli in adolescent and adult male Sprague-Dawley rats, Behav. Brain Res. 226 (2012) 613–618. [38] J. Burgdorf, B. Knutson, J. Panksepp, Anticipation of rewarding electrical brain stimulation evokes ultrasonic vocalization in rats, Behav. Neurosci. 114 (2000) 320–327. [39] S. Mitsui, M. Yamamoto, M. Nagasawa, K. Mogi, T. Kikusui, N. Ohtani, M. Ohta, Urinary oxytocin as a noninvasive biomarker of positive emotion in dogs, Horm. Behav. 60 (2011) 239–243. [40] A.S. Matthiesen, A.B. Ransjö-Arvidson, E. Nissen, K. Uvnäs-Moberg, Postpartum maternal oxytocin release by newborns: effects of infant hand massage and sucking, Birth 28 (2001) 13–19.
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
ARTICLE IN PRESS
NSL 31354 1–6
Neuroscience Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
1
Activation of hypothalamic oxytocin neurons following tactile stimuli in rats
2
3
4
Q2
Shota Okabe, Masahide Yoshida, Yuki Takayanagi, Tatsushi Onaka ∗ Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University, Shimotsuke-shi, Tochigi-ken 329-0498, Japan
5 6
h i g h l i g h t s
7 8
• Stroking stimuli activated hypothalamic paraventricular oxytocin neurons. • Rats emitted 50-kHz ultrasonic vocalizations during stroking stimuli. • Pleasant stroking is thought to activate hypothalamic oxytocin neurons.
9 10 11 12
a r t i c l e
13 28
i n f o
a b s t r a c t
14
Article history: Received 21 April 2015 Received in revised form 16 May 2015 Accepted 25 May 2015 Available online xxx
15 16 17 18 19 20
27
Keywords: Oxytocin Stroking Touch Fos protein Ultrasonic vocalizations Hypothalamus
29
1. Introduction
21 22 23 24 25 26
Gentle touching or stroking has anxiolytic actions and contributes to the establishment of an intimate relationship between individuals. Oxytocin administration also has anxiolytic actions and facilitates social behaviors. In this study, we examined effects of stroking stimuli on activation of oxytocin neurons and emission of 50-kHz ultrasonic vocalizations, an index of positive emotion, in rats. The number of oxytocin neurons expressing Fos protein was increased in the hypothalamus, especially in the dorsal zone of the medial parvicellular part of the paraventricular nucleus. The number of 50-kHz ultrasonic vocalizations was also increased. These findings suggest that pleasant sensory stimuli activate hypothalamic oxytocin neurons. © 2015 Published by Elsevier Ireland Ltd.
Gentle touching or stroking induces a pleasant sensation via activation of slow-conducting unmyelinated nerves [1,2]. Affective touch plays an important role in affiliative behaviors including parental care and pair-bonding to reduce anxiety [3], to induce analgesia [4] and to contribute to the establishment of an intimate relationship [5]. Information of this affective-motivational dimension of touch is processed in social brain regions including the medial prefrontal cortex, cingulate cortex, insula, and superior temporal sulcus [6]. However, subcortical regions that are activated by affective touch are largely unclear. Rats emit ultrasonic vocalizations dependent on the their emotional states. In aversive situations such as during predator exposure, lower frequency vocalizations, 22-kHz vocalizations, are emitted [7]. In appetitive situations such as during social investi-
30 31 32 33 34 35 36 37 38 39 40 41 42 43
Q3
∗ Corresponding author. Tel.: +81 285587318; fax: +81 285448147. E-mail address: [email protected] (T. Onaka).
gation and rough-and-tumble play, higher frequency vocalizations, 50-kHz vocalizations, are elicited [7]. Thus, 22-kHz and 50-kHz vocalizations have been used as indices of negative and positive states of rats, respectively [7]. Oxytocin, which is mainly synthesized in the hypothalamus and the bed nucleus stria terminalis (BNST), has been shown to be associated with social behaviors [8–15], to induce anti-stress or anxiolytic actions [16] and to induce analgesia [17]. Oxytocin is released from the neurohypophysis into the blood at birth and during lactation and acts on peripheral tissues. Oxytocin has also been shown to be released from dendrites or cell bodies as well as from axonal terminals of oxytocin neurons within the brain following appropriate stimuli [18]. Massage or non-noxious tactile stimulation induces an increase in plasma oxytocin [19–21]. However, the localization of oxytocin neurons activated by non-noxious sensory stimuli has remained to be identified. In this study, we examined the emission of ultrasonic vocalizations, an index of emotional states, and expression of Fos protein, an index of neuronal activation, in oxytocin neurons of the hypothalamus and BNST following tactile stimuli in rats.
http://dx.doi.org/10.1016/j.neulet.2015.05.055 0304-3940/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
G Model NSL 31354 1–6
ARTICLE IN PRESS S. Okabe et al. / Neuroscience Letters xxx (2015) xxx–xxx
2 64
2. Methods
65
2.1. Animals
76
Twenty-four male rats (9 weeks old, Iar: Long–Evans, Institute for Animal Reproduction, Ibaraki, Japan) were housed individually under a 12:12 h light/dark cycle (lights on at 7:30 am) at 22 ± 2 ◦ C and 55 ± 15% relative humidity. Food and water were available ad libitum. Animal experiments were carried out after receiving approval from the Animal Experiment Committee of Jichi Medical University and were in accordance with the Institutional Regulations for Animal Experiments and Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology.
77
2.2. Massage-like stroking
66 67 68 69 70 71 72 73 74 75
87
Rats were placed on the experimenter’s lap and given stroking stimuli on the dorsal part of their bodies by the experimenter’s hand with a cotton glove at a speed of 8.76 ± 0.17 cm/s (mean ± SEM, n = 8) and a frequency of 0.62 ± 0.01 Hz (mean ± SEM, n = 8) for 5 min without immobilization of their bodies. The speed and frequency of the stroking stimuli were monitored by use of a video camera. The force applied by the manual-stroking stimuli was relatively mild and was estimated to be less than 250 mN/cm2 . Cotton gloves were used for stroking to reduce friction and to make stroking as smooth as possible.
88
2.3. Recording and analysis of ultrasonic vocalizations
78 79 80 81 82 83 84 85 86
89 90 91 92 93 94 95 96 97 98 99 100 101 102
103 104
105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121
Ultrasonic vocalizations recording was performed by use of a 1/4-inch measurement-grade microphone (Type 4158N, Aco, Tokyo, Japan) designed for measurements of sound pressure levels between 20 Hz and 100,000 Hz. The microphone was placed at a distance of approximately 30 cm from the rats and was connected to a sensor amplifier (SR-2200, Ono Sokki, Kanagawa, Japan) and an AD–DA converter (USB-6251, NI, Texas, USA). Acoustic data were recorded at a sampling rate of 200 kHz and with 16 bits by an Avisoft RECORDER (Version 4.2, Avisoft Bioacoustics, Berlin, Germany) and analyzed by Avisoft SASLab Pro (Version 5.2, Avisoft Bioacoustics). Spectrograms were generated with a fast Fourier transform length of 256 points and an overlap of 50%. The numbers of 50kHz and 22-kHz vocalizations were counted manually according to previous reports [22,23]. 2.4. Immunohistochemical detection of fos protein in oxytocin-immunoreactive (-ir) neurons Ninety minutes after termination of stroking or holding stimuli, the rats were anesthetized with Avertin (tribromoethanol; 200 mg/kg, i.p.) and perfused transcardially with heparinized saline (2 U/mL) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min. The brains were immediately removed from the skulls, post-fixed in 4% paraformaldehyde overnight, placed in 30% sucrose in 0.1 M phosphate buffer until they sank, and frozen in dry ice. The hypothalamic part of each frozen brain was sectioned coronally at 30 m and processed for immunochemical detection of Fos protein and oxytocin as described previously with modifications [24]. In brief, sections were incubated with a rabbit polyclonal antibody raised against the Fos peptide sequence (Ab-5; Oncogene Science; Cambridge, MA, USA) diluted at 1:10,000 for 2 days at 4 ◦ C. Immunoreactivity was visualized by sequential overnight incubation with peroxidase-labeled goat anti-rabbit IgG (diluted at 1:500; Vector Laboratories, Inc., Burlingame, CA, USA) at 4 ◦ C and 3,3-diaminobenzidine tetrahydrochloride (DAB)
Fig. 1. Ultrasonic vocalizations during stroking and holding stimuli. A spectrogram of 50-kHz vocalizations in stroking group (A) and the numbers of 50-kHz and 22-kHz vocalizations during stroking (B) and holding (C) are shown. Rats emitted 50-kHz ultrasonic vocalizations more often than 22-kHz vocalizations during stroking and holding stimuli. *, P < 0.05. n = 8 per group.
with nickel sulphate. The sections were then processed for detection of oxytocin using an antibody against oxytocin (diluted at 1:1,000,000; Peninsula Laboratories International, San Carlos, CA, USA). After two days of incubation at 4 ◦ C with the first antibody, sections were incubated with biotinylated anti-guinea pig IgG (diluted at 1:750; Vector Laboratories) for 2 h at room temperature and with avidin biotinylated horseradish peroxidase complex (diluted at 1:50; Vector Laboratories) for 30 min. Oxytocin was visualized with DAB. Sections containing hypothalamic paraventricular nuclei (PVN) (14 sections), supraoptic nuclei (SON) (15 sections) or BNST (8 sections) were examined at intervals of 120 m in each rat. Areas of the brain were identified according to brain atlases [25,26] 2.5. Groups and experimental design All rats received 5-min stroking stimuli once a day for 7 days. On the following day, rats were assigned to one of three groups: stroking group (n = 8), holding group (n = 8), and non-touch group (n = 8). In the stroking group, rats were placed on the experimenter’s lap and given massage-like stroking for 5 min. In the holding group, rats were handled in the same manner as rats in the stroking group for 5 min without receiving stroking stimuli. In the non-touch group, rats were kept in their individual home cages. In the stroking and holding groups, ultrasonic vocalizations were monitored by use of an ultrasound microphone. After 5-min manipulation, rats were returned to their home cages. Ninety minutes after termination of stroking or holding stimuli, the rats were perfused with 4% paraformaldehyde following deep anesthesia. 2.6. Statistical analysis Data are expressed as means ± SEM. The numbers of 50-kHz and 22-kHz ultrasonic vocalizations were analyzed by Wilcoxon’s signed rank test or the Mann–Whitney U test. The number of immunoreactive cells was analyzed by one-way ANOVA followed
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
122 123 124 125 126 127 128 129 130 131 132 133 134
135
136 137 138 139 140 141 142 143 144 145 146 147 148 149
150
151 152 153 154
G Model NSL 31354 1–6
ARTICLE IN PRESS S. Okabe et al. / Neuroscience Letters xxx (2015) xxx–xxx
3
Fig. 2. Expression of Fos protein in oxytocin-immunoreactive (-ir) neurons following stroking stimuli. Oxytocin-ir neurons expressing Fos protein in the bed nucleus of the stria terminalis (BNST) (A1, B1, C1), the supraoptic nucleus (SON) (A2, B2, C2), and the paraventricular nucleus (PVN) (A3, B3, C3) in the stroking (A1–A3), holding (B1-B3), and non-touch groups (C1–C3) and the percentages of oxytocin-ir neurons expressing Fos protein in the BNST (D), SON (E), and PVN (F) are shown. The percentages of oxytocin-ir neurons expressing Fos protein were significantly increased in the SON and PVN after stroking as compared with the non-touch group. Brown cytoplasmic reactions indicate oxytocin immunoreactivity, and dark nuclear reactions indicate Fos immunoreactivity. Areas enclosed by black rectangles in A2 and A3 are shown at a higher magnification. The arrowheads indicate double-labeled neurons. Scale bar in A1 = 50 m. Scale bar in black rectangles = 12.5 m. *, P < 0.05. n = 8 per group.
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
G Model NSL 31354 1–6
ARTICLE IN PRESS S. Okabe et al. / Neuroscience Letters xxx (2015) xxx–xxx
4
Fig. 3. Expression of Fos protein in oxytocin-ir neurons in the dorsal zone of the medial parvicellular part of the PVN following stroking stimuli. Oxytocin-ir neurons expressing Fos protein in the dorsal zone of the medial parvicellular part of the PVN in the stroking (A), holding (B), and non-touch groups (C), and the percentages of oxytocin-ir neurons expressing Fos protein in the dorsal zone of the medial parvicellular part of the PVN (D) are shown. The percentage of oxytocin-ir cells expressing Fos protein was significantly increased after stroking as compared with non-touch or holding group. The arrowhead indicates a double-labeled neuron. Scale bar = 5 m. *, P < 0.05. n = 8 per group.
156
by the Tukey–Kramer post hoc test. P < 0.05 was considered statistically significant.
more than that of 22-kHz vocalizations in both groups (Fig. 1B and C; P < 0.05, Wilcoxon’s signed rank test).
157
3. Results
3.2. Expression of Fos protein in oxytocin-immunoreactive (-ir) neurons
158
3.1. Ultrasonic vocalizations
155
159 160 161
In the stroking group, rats emitted 50-kHz vocalizations (Fig. 1A). In addition, in the holding group, rats emitted 50-kHz vocalizations. The number of 50-kHz vocalizations was significantly
In the BNST, SON and PVN, there were no significant differences in the total number of oxytocin-ir cells or Fos protein-ir cells among the stroking, holding and non-touch control groups (data not shown). However, compared to those in the non-touch control group, the percentages of oxytocin-ir cells expressing Fos protein
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
162 163
164 165
166 167 168 169 170
G Model NSL 31354 1–6
ARTICLE IN PRESS S. Okabe et al. / Neuroscience Letters xxx (2015) xxx–xxx
171 172 173 174 175 176 177 178 179
180
181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233
in the stroking group were significantly higher in the SON (Fig. 2A2, B2, C2, and E; P < 0.05) and PVN (Fig. 2A3, B3, C3, and F; P < 0.05) but not in the BNST (Fig. 2A1, B1, C1, and D). Oxytocin-ir cells expressing Fos protein were observed in the PVN, especially in the dorsal zone of the medial parvicellular part. Compared to those in the non-touch control and holding group, the percentages oxytocin-ir cells expressing Fos protein in the stroking group were significantly higher in the dorsal zone of the medial parvicellular part (Fig. 3; P < 0.05).
4. Discussion Tactile stimuli or massage have been shown to increase plasma oxytocin concentrations in humans [27]. In the present study, we demonstrated that gentle stroking activated oxytocin neurons in PVN and SON neurons of rats. During stroking stimuli, the animals emitted 50-kHz ultrasonic vocalizations, which have been associated with positive states [7]. The findings suggest that affective sensory stimuli activate hypothalamic oxytocin neurons. The present study showed that gentle stroking induced expression of Fos protein, an index of neuronal activation, in oxytocin neurons of the hypothalamic PVN and SON. Plasma oxytocin concentrations were not measured in the present study. However, following one-minute stroking stimuli applied to the whole bodies of rats held by the experimenter’s hand, plasma oxytocin concentrations were slightly but significantly increased (Wistar strain, 11.6 ± 0.9 pg/ml for the control group, 14.5 ± 0.8 pg/ml for the stroking group, n = 6, P < 0.05, t-test, unpublished data). Although the procedures used for stroking stimuli were slightly different from the present study (held by hand vs free-moving, applied to the whole body vs the back, Wistar strain vs Long–Evans strain), it is likely that oxytocin release into plasma might also have been facilitated in response to non-noxious tactile stimuli in the present study. In the present study, oxytocin neurons activated following tactile stimuli were mainly located in the dorsal zone of the medial parvicellular part of the PVN (Fig. 3). Oxytocin neurons in the PVN have been shown to project to the ventral tegmental areas, periaqueductal gray, nucleus tractus solitarius, and spinal cord [28,29,30]. These brain areas are associated with processing for reward [31], pain [32], and cardiovascular control [33]. Both oxytocin application and non-noxious sensory stimuli modulate positive emotional behavior [3,21], induce analgesia [17,20], facilitate affiliative behavior [5,34], and change cardiovascular parameters [35,36]. It is thus interesting to speculate that oxytocin is involved in these actions of non-noxious sensory stimulation by acting on the ventral tegmental areas, periaqueductal gray, nucleus tractus solitarius, or spinal cord. Further research is necessary to clarify the physiological roles of oxytocin in effects of tactile stimuli. In the present study, rats emitted 50-kHz vocalizations while being held without receiving stroking as well as during stroking stimuli. Emission of 50 kHz vocalizations is considered to reflect positive emotion [7]. The animals in the present study received stroking stimuli while being held for 7 days, and 50-kHz ultrasonic vocalizations have been shown to be emitted not only during affiliative social contact but also during anticipation of social contact [37] or reward anticipation [38]. It is thus possible that holding stimuli might have induced anticipation of stroking stimuli and induced 50-kHz ultrasonic vocalizations in the present study. Oxytocin neurons are activated by various kinds of stressful stimuli [13]. The data obtained in this study showing that stroking stimuli activated oxytocin neurons in the PVN might be explained by stress due to stroking stimuli. However, it is not likely. In the present study, animals emitted 50-kHz ultrasonic vocalizations during stroking. On the other hand, the number of
5
22-kHz ultrasonic vocalizations, which reflect negative emotions [7], was significantly less than that of 50-kHz ultrasonic vocalizations during stroking stimuli. Consistent with the idea that stroking stimuli were not stressful in the present study, the number of Fos-positive paraventricular non-oxytocin neurons, which are thought to include CRF-synthesizing neurons, was not significantly increased following stroking stimuli (data not shown). Non-noxious sensory stimuli have been shown to increase plasma or urinary oxytocin concentrations in rats [21], dogs [39], and humans [27]. Massage-like hand movement of a baby induces an increase in plasma oxytocin concentration in the mother [40]. From all these data, we conclude that affiliative tactile stimuli activate oxytocin neurons in the hypothalamus. Acknowledgments This work was supported in part by grants from the Japan Soci- Q4 ety for the Promotion of Science (15K18978 to S.O.,26460322 to Y.T., 26460321 to M.Y. and 25118008, 26293049 and 15K15042 to T.O.,) and by the the MEXT (the Ministry of Education, Culture, Sports, Science and Technology of Japan -Supported Program for the Strategic Research Foundation at Private Universities, 2011-2015 (Cooperative Basic and Clinical Research on Circadian Medicine) References [1] H. Olausson, Y. Lamarre, H. Backlund, C. Morin, B.G. Wallin, G. Starck, S. Ekholm, I. Strigo, K. Worsley, Å.B. Vallbo, M.C. Bushnell, Unmyelinated tactile afferents signal touch and project to insular cortex, Nat. Neurosci. 5 (2002) 900–904. [2] L.S. Löken, J. Wessberg, I. Morrison, F. McGlone, H. Olausson, Coding of pleasant touch by unmyelinated afferents in humans, Nat. Neurosci. 12 (2009) 547–548. [3] A. Gallace, C. Spence, The science of interpersonal touch: an overview, Neurosci. Biobehav. Rev. 34 (2010) 246–259.
234 235 236 237 238 239 240 241 242 243 244 245 246
247
248 249 250 251 252 253 254
255
256 257 258 259 260 261 262 263 264 265
[4] G. Agren, T. Lundeberg, K. Uvnäs-Moberg, A. Sato, The oxytocin antagonist 1-deamino-2-D-tyr-(oet)-4-thr-8-orn-oxytocin reverses the increase in the withdrawal response latency to thermal, but not mechanical nociceptive stimuli following oxytocin administration or massage-like stroking in rats, Neurosci. Lett. 187 (1995) 49–52. [5] R.I.M. Dunbar, The social role of touch in humans and primates: behavioural function and neurobiological mechanisms, Neurosci. Biobehav. Rev. 34 (2010) 260–268. [6] I. Gordon, A.C. Voos, R.H. Bennett, D.Z. Bolling, K.A. Pelphrey, M.D. Kaiser, Brain mechanisms for processing affective touch, Hum. Brain Mapp. 34 (2013) 914–922. [7] S.M. Brudzynski, Ethotransmission: communication of emotional states through ultrasonic vocalization in rats, Curr. Opin. Neurobiol. 23 (2013) 310–317. [8] Z.R. Donaldson, L.J. Young, Oxytocin, vasopressin, and the neurogenetics of sociality, Science 7 (2008) 900–904. [9] A.M. Kelly, J.L. Goodson, Social functions of individual vasopressin-oxytocin cell groups in vertebrates: what do we really know? Front. Neuroendocrinol. 35 (2014) 512–529. [10] R. Stoop, Neuromodulation by oxytocin and vasopressin in the central nervous system as a basis for their rapid behavioral effects, Curr. Opin. Neurobiol. 29 (2014) 187–193. [11] C.S. Carter, Oxytocin pathways and the evolution of human behavior, Annu. Rev. Psychol. 65 (2014) 17–39. [12] H.S. Knobloch, V. Grinevich, Evolution of oxytocin pathways in the brain of vertebrates, Front. Behav. Neurosci. 8 (2014) 31. [13] T. Onaka, Y. Takayanagi, M. Yoshida, Roles of oxytocin neurones in the control of stress energy metabolism, and social behaviour, J. Neuroendocrinol. 24 (2012) 587–598. [14] H. Higashida, S. Yokoyama, M. Kikuchi, T. Munesue, CD38 and its role in oxytocin secretion and social behavior, Horm. Behav. 61 (2012) 351–358. [15] M. Nagasawa, S. Mitsui, S. En, N. Ohtani, M. Ohta, Y. Sakuma, T. Onaka, K. Mogi, T. Kikusui, Oxytocin-gaze positive loop and the coevolution of human-dog bonds, Science 348 (2015) 333–336. [16] M. Yoshida, Y. Takayanagi, K. Inoue, T. Kimura, L.J. Young, T. Onaka, K. Nishimori, Evidence that oxytocin exerts anxiolytic effects via oxytocin receptor expressed in serotonergic neurons in mice, J. Neurosci. 29 (2009) 2259–2271. [17] M. Petersson, P. Alster, T. Lundeberg, K. Uvnäs-Moberg, Oxytocin increases nociceptive thresholds in a long-term perspective in female and male rats, Neurosci. Lett. 212 (1996) 87–90.
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306
G Model NSL 31354 1–6 6 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340
ARTICLE IN PRESS S. Okabe et al. / Neuroscience Letters xxx (2015) xxx–xxx
[18] G. Leng, M. Ludwig, Neurotransmitters and peptides: whispered secrets and public announcements, J. Physiol. 586 (2008) 5625–5632. [19] S. Stock, K. Uvnäs-Moberg, Increased plasma levels of oxytocin in response to afferent electrical stimulation of the sciatic and vagal nerves and in response to touch and pinch in anaesthetized rats, Acta. Physiol. Scand. 132 (1988) 29–34. [20] K. Uvnäs-Moberg, G. Bruzelius, P. Alster, T. Lundeberg, The antinociceptive effect of non-noxious sensory stimulation is mediated partly through oxytocinergic mechanisms, Acta. Physiol. Scand. 149 (1993) 199–204. [21] K. Uvnäs-Moberg, L. Handlin, M. Petersson, Self-soothing behaviors with particular reference to oxytocin release induced by non-noxious sensory stimulation, Front. Psychol. 5 (2015) 1529. [22] S.M. Brudzynski, Pharmacological and behavioral characteristics of 22khz alarm calls in rats, Neurosci. Biobehav. Rev. 25 (2001) 611–617. [23] J. Burgdorf, R.A. Kroes, J.R. Moskal, J.G. Pfaus, S.M. Brudzynski, J. Panksepp, Ultrasonic vocalizations of rats (Rattus norvegicus) during mating, play, and aggression: behavioral concomitants, relationship to reward, and self-administration of playback, J. Comp. Psychol. 122 (2008) 357–367. [24] L. Zhu, T. Onaka, Involvement of medullary A2 noradrenergic neurons in the activation of oxytocin neurons after conditioned fear stimuli, Eur. J. Neurosci. 16 (2002) 2186–2198. [25] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, sixth ed., Academic Press, San Diego, 2007. [26] L.W. Swanson, Brain Maps: Structure Of The Rat Brain, second ed., Elsevier, Amsterdam, 1999. [27] V. Morhenn, L.E. Beavin, P.J. Zak, Massage increases oxytocin and reduces adrenocorticotropin hormone in humans, Altern. Ther. Health Med. 18 (2012) 11–18. [28] M.V. Sofroniew, Projections from vasopressin oxytocin, and neurophysin neurons to neural targets in the rat and human, J. Histochem. Cytochem. 28 (1980) 475–478. [29] T.A.P. Roeling, J.G. Veening, J.P.W. Peters, M.E.J. Vermelis, R. Nieuwenhuys, Efferent connections of the hypothalamic grooming area in the rat, Neuroscience 56 (1993) 199–225.
[30] J.D. Breton, P. Veinante, S. Uhl-Bronner, A.M. Vergnano, M.J. Freund-Mercier, R. Schlichter, P. Poisbeau, Oxytocin-induced antinociception in the spinal cord is mediated by a subpopulation of glutamatergic neurons in lamina I-II which amplify GABAergic inhibition, Mol. Pain 4 (2008). [31] S. Lammel, B.K. Lim, R.C. Malenka, Reward and aversion in a heterogeneous midbrain dopamine system, Neuropharmacology 76 (2014) 351–359. [32] M.M. Heinricher, I. Tavares, J.L. Leith, B.M. Lumb, Descending control of nociception: specificity, recruitment and plasticity, Brain Res. Rev. 60 (2009) 214–225. [33] A. Krassioukov, V.E. Claydon, The clinical problems in cardiovascular control following spinal cord injury: an overview, Prog. Brain Res. 152 (2005) 223–229. [34] T.R. Insel, The challenge of translation in social neuroscience: a review of oxytocin, vasopressin, and affiliative behavior, Neuron 65 (2010) 768–779. [35] M. Petersson, P. Alster, T. Lundeberg, K. Uvnäs-Moberg, Oxytocin causes a long-term decrease of blood pressure in female and male rats, Physiol. Behav. 60 (1996) 1311–1315. [36] M. Kurosawa, T. Lundeberg, G. Ågren, I. Lund, K. Uvnäs-Moberg, Massage-like stroking of the abdomen lowers blood pressure in anesthetized rats: influence of oxytocin, J. Auton. Nerv. Syst. 56 (1995) 26–30. [37] A.R. Willey, L.P. Spear, Development of anticipatory 50 kHz usv production to a social stimuli in adolescent and adult male Sprague-Dawley rats, Behav. Brain Res. 226 (2012) 613–618. [38] J. Burgdorf, B. Knutson, J. Panksepp, Anticipation of rewarding electrical brain stimulation evokes ultrasonic vocalization in rats, Behav. Neurosci. 114 (2000) 320–327. [39] S. Mitsui, M. Yamamoto, M. Nagasawa, K. Mogi, T. Kikusui, N. Ohtani, M. Ohta, Urinary oxytocin as a noninvasive biomarker of positive emotion in dogs, Horm. Behav. 60 (2011) 239–243. [40] A.S. Matthiesen, A.B. Ransjö-Arvidson, E. Nissen, K. Uvnäs-Moberg, Postpartum maternal oxytocin release by newborns: effects of infant hand massage and sucking, Birth 28 (2001) 13–19.
Please cite this article in press as: S. Okabe, et al., Activation of hypothalamic oxytocin neurons following tactile stimuli in rats, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.055
341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
