Ayako Homma, BA.1,2, Hideki Hara, PhD 1,3, Kumiko Matsuzaki, PhD 2, Yoshiko Nakagawa, PhD1, Yuri Masaoka, PhD1 and Ikuo Homma, MD, PhD1

1 Department of Physiology, Showa University School of Medicine, Tokyo Japan
2 Department of Pediatrics, Showa University School of Medicine, Tokyo Japan
3 Faculty of Letters, Kokugakuin University, Tokyo Japan

Corresponding author: Ikuo Homma, Department of Physiology, Show University School of Medicine, Hatanodai 1-5, Shinagawa-ku, Tokyo 142-8555, Japan
e-mail: ihomma@med.showa-u.ac.jp    tel: +81-3-3784-8112, fax: +81-3-3784-0200



Abstract

The touching of animals as therapy is widely accepted in various medical applications. Dolphin facilitated therapy has been applied in the treatment of autistic individuals and in adult and child patients with depression. However, there are no studies on the effectiveness of this therapy with respect to psychological states as correlated with physiologic states or focal brain activity.  There are many reports that respiration is altered by various emotions and may be an index for the measurement of certain emotional states in humans. In this study, we examined the effects of dolphin touching on the emotional state or anxiety, respiratory rate, and brain activity in seven children (7-12 years old). State anxiety scores were significantly decreased, and respiratory rate was also decreased after dolphin touching. EEG frequency was used as index for investigating individuals in awake and arousal state. Frequencies between 4 to 12 Hz (theta and alpha) were defined as a relaxed state and frequencies between 13 to 30 Hz (beta) as an arousal state.

Beta waves were significantly increased in the anxiety state, but decreased after touching dolphins. Previously we found that respiratory rate increased during anxious states, and we determined that activity in the amygdale is correlated both with increase in respiration and anxiety. The dipole tracing method enables detection of the source of the event-related potentials. In this study, onset of inspiration before dolphin touching were used for averaging the EEG. We found that a negative EEG potential was observed 150-250 msec after the onset of inspiration. Dipoles were located in the amygdala, insula, and temporal pole during this period. However, this potential was not observed after touching. These results suggest higher levels of anxiety before dolphin touching, which were decreased after dolphin touching.

Key words: dolphin, respiration, anxiety, brain, EEG, amygdala


Introduction

The aim of the present study was to examine how children’s anxiety scores, breathing patterns, EEG activity, and brain activity synchronized with breathing are affected by dolphin-touching. We hypothesized that dolphin touching affects emotion and respiration-related brain activity, resulting in a relaxed state.

Effects of animals on healing in humans have attracted widespread interest. Some animal-facilitated therapies are thought to contribute to the treatment of certain disorders. For example, patients with heart failure who are pet owners have higher survival rates than those who are not pet owners (Friedmann et al. 1980). It has been reported that dog owners are motivated to take daily walks and respond to stress more effectively (Rogers et al. 1993). The psychological efficacy of animal facilitated therapy is reflected in changes in autonomic nervous system activity, which affects circulation and respiration. Living with a pet decreases blood pressure and stabilizes heart rate (Katcher et al. 1983), and pets are integral parts of the social support network for humans. There are several anecdotal reports of the beneficial effects of dogs, cats and horses in animal facilitated therapy as mention above. However, there are few reports on dolphin-facilitated therapy, despite the high level of general interest in dolphins (Antonioli and Reverley, 2005; Smith, 1987). Smith, a pioneer in dolphin-facilitated therapy, has shown desirable effects of this therapy on behavior, emotion, and speech ability in autistic individuals (Smith, 1987). Recently, the efficacy of dolphin-facilitated therapy was reported in adult patients with depression; symptoms of depression were alleviated after two weeks of therapy (Antonioli and Reverley, 2005).

The major effect of animal therapy is on subjective affect and emotion. However, there have been a few studies showing effects on psychological and behavioral parameters and focal brain activity. Psychophysiological studies have focused on relations between emotions and physiological changes and have shown mirrored effects on feelings and thoughts. For example, skin conductance response, heart rate and respiration change during fear and anxiety (Hugdahl, 1966; Davis, 1992). Emotional changes are accompanied by changes in the autonomic nervous system, which affect heart rate and respiration. Breathing is regulated in the brainstem primarily for metabolic purposes. In the awake state, breathing patterns are regulated by a complex interaction between metabolic and non-homeostatic requirements. Breathing in response to emotional arousal is a component of the non-homeostatic system. Respiratory rate increases with increased level of anxiety (Masaoka and I Homma, 1999) and correlates with individual anxiety scores (Masaoka and I Homma, 2001).

Electroencephalography (EEG) is noninvasive and therefore has an advantage that it can be used to examine brain activity related to emotion. There are many studies investigating psychophysiological changes with frequency analysis of or waveforms.  De Bergerac (2005) reported that theta and alpha waves increased in dolphin-facilitated therapy. Neuroanatomic correlates of various emotions have been investigated in humans by positron emission tomography and functional MRI, which show increases in local blood flow and hemoglobin concentration, corresponding to local neuronal activity in the brain (Lane et al. 1997; Reiman, et al. 1989). EEG dipole tracing (DT) is a neuroimaging method that can determine focal areas of neuronal activation triggered by various tasks. The development of DT methods has progressed recently, and Homma et al. have incorporated a realistic three-shell head model (Scalp-Skull-Brain; SSB/DT) that can locate dipoles with high spatial resolution (Homma et al. 1994; Homma and Masaoka et al. 2001).


Methods

Subjects
Seven healthy children aged 7-10 years (mean age, 9.3 ± 1.2 years) without no psychiatric disorder participated in this study. All subjects were naïve to the purpose of the experiment, and informed consent was obtained from each subject and his or her parent. The study was approved by the Ethics Committee of Showa University School of Medicine. The study was performed at a dolphin pool in the bay of Ito City, Shizuoka prefecture, Japan (Dolphin Fantasy). The equipment for the study was set up in a room beside the pool. The children wore waterproof suits and were guided to the edge of the pool by a dolphin trainer. They sat at the edge of the pool with their legs in the water. They were asked to touch the dolphin with their hands under the direction of the dolphin trainer. Their parents sat on the pool side away from the trainer and subjects

Assessment of psychological state and respiration
Before moving to the edge of pool, subjects’ anxiety levels were evaluated by Spielberger’s State-Trait Anxiety Inventory (STAI) (Spielberger, 1983). The STAI was modified for children and translated into Japanese (Sankyoubou, Kyoto, Japan). The STAI is self-administered and consists of two anxiety scores, trait and state. Each test consists of 20 items. The trait anxiety score is used to assess how the individual feels generally and is not usually influenced by any condition. The state anxiety score is used to evaluate how the individual feels at the current moment in a given situation. The state anxiety score was also obtained after dolphin touching while resting by the pool.  Anxiety levels of all subjects except one were evaluated before and after dolphin touching. One subject, who was 6 years old, was excluded because he misunderstood the questions.

Respiratory movements were measured continuously with a band with an induction coil mounted around the chest (TR-112A; Nihon Koden, Tokyo Japan). Data were sent wirelessly from a transmitter attached to the lumbar region of the subject to a receiver in the room by the pool. Data were stored by an EEG analyzer (DAE-2; Nihon Koden, Tokyo, Japan). Respiratory rate before and after dolphin touching was calculated from the stored data. Respiratory movement was used as the trigger signal for EEG averaging.

EEG
An Electro-Cap was used with nineteen silver electrode placement according to the international 10-20 system (Nihon Koden). Electrodes were attached to the scalp of the children, and reference electrodes were placed bilaterally on the earlobes. Electro-gel (Nihon Koden) was used with each electrode. Two additional electrodes were attached to the face near the eyes to measure ocular activity. EEG and electro-oculogram signals were sent wirelessly from a transmitter to a receiver and triggered by respiratory movement. Data were stored in a digital EEG analyzer. EEGs were sampled at 200 Hz through a 0.016-30 Hz bandpass filter. Electrode impedance was kept below 10 kΩ. After attachment of electrodes and other apparatus, the subjects moved to the side of the pool and sat down.

DT analysis
The onset of inspiration was signaled by respiratory movement, which was used as a trigger for averaging EEG potentials. Pre 100 and post 600 ms-inspiration onsets were averaged. Averaged potentials were assessed before and after dolphin touching. Eye blinks and artifacts exceeding ±50 v were excluded. The EEG data could include artifacts due to environmental noise or background activity. Potentials were averaged across the six subjects to enhance the signal-to-noise ratio. Averaged EEG potentials were transferred to an SSB/DT system (Brain Space navigator, BS-Navi; Brain Research and Development, Tokyo, Japan) for analysis. Details of the SSB/DT method are reported elsewhere (Homma et al. 1994; Musha and Okamoto, 1999). SSB/DT calculates the source distribution, which is approximated by equivalent current dipoles. The actual potential distributions recorded from the scalp electrodes were compared to the calculated distribution of equivalent dipoles. We used the inverse solution, which assesses three-dimensional electrical brain activity via two-dimensional surface potentials (He et al. 1987). The locations of one or two current dipoles within the head model were changed by the simplex method to obtain the minimum square difference between the actual potentials and calculated potentials (Kowalik and Osborne, 1968). The degree of source concentration can be calculated in terms of dipolarity. In the present study, dipolarity greater than 98% was used to indicate source concentration (Masaoka and Homma, 2001; Homma et al. 2001; Ikeda et al. 1998; Kanamaru et al. 1999; Masaoka et al. 2005). Dipole calculations were performed with three layers. The scalp-skull-brain head model created from the standard MNI template, was downloaded from the Cognition, Emotion and Psychopathology Program homepage (http://imaging.mrc-cbu.cam.ac.uk). Conductivities of the brain (0.33 S/m), skull (0.0041 S/m), and scalp (0.33 S/m) were incorporated into the calculations.

EEG power spectrum analysis
The EEG power of the band component were calculated over 700 ms of mean values of the six subjects. Spectral power was analyzed by fast Fourier transformation (spectral analysis) with EEG analysis software (EEG Focus, Version 2.1; Nihon Kohden, Tokyo, Japan).

Data analysis
All statistical analyses were performed with a commercially available statistical package (SPSS, Version 11.00; SPSS Tokyo, Japan). State anxiety score, respiratory rate, and the power spectrum before and after dolphin touching were analyzed by one-way repeated measures analysis of variance (ANOVA). P values < 0.05 were considered significant.


Results

TRAIT and STATE scores
Subject, sex, age, TRAIT anxiety score, and STATE anxiety score before (Pre-) and after (Post-) dolphin touching are listed in Table 1. The mean TRAIT anxiety score (mean ± SD) was 31.3 ± 7.34, the mean Pre STATE score was 35.8 ± 6.05, and the mean Post STATE score was 29.5 ± 7.06.

The mean Pre-touching State Anxiety scores were significantly greater than the mean Post-touching State Anxiety scores (P ‹ 0.05) (Figure 1).



Respiratory rate
The mean Pre respiratory rate of all six subjects was 22.3 ± 1.40 breaths/min, and the mean Post rate was 20.8 ± 3.72 breaths/min. The Pre respiratory rate was significantly greater than the Post rate (P ‹ 0.05) (Figure 2).

Power spectra
Representative examples of power spectra for the 19 electrodes are shown in Figure 3. For each electrode, the power of high bands was greater pre- versus post- dolphin touching. Mean power values for theta and alpha (4-12 Hz) and beta (13-30 Hz) waves for each electrode were compared in all subjects. Mean power percentages of positive and negative waves at the C4 electrode pre- and post- dolphin touching are shown in Figure 4. The mean power percentage of theta and alpha wave for all six subjects was 56.5 ± 2.6 pre and 61.7 ± 4.7 post dolphin touching. The mean power percentage of the beta wave for all six subjects was 43.3± 2.3 pre and 38.2 ± 4.6 post dolphin touching. The theta and alpha wave pre- value was significantly lower than the post- value (P ‹ 0.01), and the beta wave pre- value was significantly greater than the post- value (P ‹ 0.01).

The y-axis indicates power percentage.
(A) theta and alpha wave percentages before and after touching.
(B) Beta wave percentages before and after touching.
The power of the theta and alpha waves was significantly
greater after touching compared to before touching (P ‹ 0.01).
Values are mean ± SD.

Inspiratory phase-locked source generators
The grand-averaged EEG of the 19 channels triggered at the onset of inspiration is shown in Figure 5A. Before dolphin touching, a negative potential from 150 to 250 msec after the onset of inspiration was observed. Root mean square values (RMS) show the mean potentials of absolute values of the 19 channels. These values were high from 150 to 250 msec after the onset of inspiration. Dipoles with dipolarity 98% were superimposed on MNI brain images (Figure 5B). Dipoles were observed in the left amygdala 165-180 msec after the onset of inspiration in both horizontal and coronal sections. At the peak of the negative wave, 200 msec after the onset of inspiration, dipoles were observed in the insula. At 225 msec after the onset of inspiration, dipoles were observed in the temporal pole.


 
Discussion

In the present study, we found that respiratory rate and anxiety level were decreased after dolphin touching than those before touching.  Thus state anxiety scores as well as physiologic responses reflect a relaxed state after dolphin touching in children.

There are many reports showing that animal therapy has beneficial psychophysiological effects on humans. We studied healthy children and showed that their state anxiety scores decreased after dolphin touching. The mean STATE score before dolphin touching was 35.8, which was in the normal range (33-43). Therefore their anxiety was not high before dolphin touching. However, the mean score after dolphin touching was 29.5, which was in the low range (20-32) and was significantly lower than the mean Pre value.

Emotions are reflected in autonomic nervous system activity, which influence heart rate and respiration (Boiten et al. 1994). Respiratory rate increases during anticipatory anxiety and increased respiratory rate is positively correlated with trait anxiety scores (Masaoka and Homma, 1999; Masaoka and Homma, 2001).

In addition to decreased STATE anxiety scores and decreased respiratory rate after dolphin touching, power spectrum analysis showed that theta and alpha waves increased from 56.6% to 61.7%. However, beta waves decreased from 43.3% to 38.2%. Increased theta and alpha waves reflect decreased anxiety, and this has been used in assessing results of therapy for alcohol abuse and psychological trauma (De Bergerac, 2005). An increase in theta and alpha waves may be related to a relaxed state and could be an index for measuring anxiety level and physiologic state.

It is interesting to note that alpha waves are synchronized with inspiration during olfactory stimulation in humans (Masaoka et al. 2005). This is referred as inspiration phase-locked alpha band oscillation (I-alpha). In the present study, theta and alpha, and beta waves were synchronized with inspiration, and this wave generation was localized in the amygdala. The nature and import of this synchronization is unclear. EEG rhythm may be synchronized with respiratory rhythm. Recently, Onimaru and Homma (2007) reported respiratory rhythmic neural activity in the amygdala in the limbic-brainstem-spinal cord preparation of newborn rats. The respiratory rhythm was generated in the piriform cortex, which includes the olfactory center. Slow waves generated in the piriform cortex are thought to be necessary the development of the nervous system. I-alpha during olfactory stimulation in humans may correspond to respiratory rhythm recorded in animal experiments.


DT analysis

In the present study, we recorded a negative wave from 150 to 250 msec after the onset of inspiration. In the early phase in the wave, dipole sources were observed in the amygdala. Around the peak of the negative wave, dipole sources were observed in the insula, and in the late phase, dipole sources were observed in the temporal pole. Masaoka and Homma (2001) reported a positive wave from 200 to 400 msec after the onset of inspiration during anticipatory anxiety, referred to as the respiration-related anxiety potential (RAP). Source generators for RAPs, as determined by dipole tracing, were observed in the temporal pole and amygdala. Activated areas in the present study were observed in similar areas. The limbic and paralimbic systems particularly the amygdala, may generate activity related to respiration related emotions. These areas of activation were not observed after dolphin touching, and this reflected decreases in state anxiety and respiration rate. We suggest that dolphin-facilitated therapy could be effective for people with high anxiety or tendency to be nervous as we observed improvements in both emotional and physiological responses that correlated with changes in levels of anxiety.    

It should be noted that we did not investigate the effect of animal facilitated therapy on other emotional and physiological responses.  In future studies, we will focus on dolphin-facilitated therapy in various emotional and physiological measurements comparing the responses in other animal facilitated therapy.   


Acknowledgements
We are grateful to Mr. Hiromi Tsukuda, Mayor of Ito City, and Mr. Katsuyuki Goto, Managing Director of the Non Profit Organization of Experience Activity Meeting for their support of this study.


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