Different Locomotor Activities and Monoamine Levels in the Brains of Djungarian Hamsters (D. sungorus) and Roborovskii Hamsters (D.

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1 Exp. Anim. 57(5), , 2008 Different Locomotor Activities and Monoamine Levels in the Brains of Djungarian Hamsters (D. sungorus) and Roborovskii Hamsters (D. roborovskii) Yusuke KABUKI, Haruka YAMANE, Kousuke HAMASU, and Mitsuhiro FURUSE Laboratory of Advanced Animal and Marine Bioresources, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka , Japan Abstract: Two species of the genus Phodopus, Djungarian hamster (P. sungorus) and Roborovskii hamster (P. roborovskii), differ in their behavior. The Roborovskii hamster has high locomotor activity (hyperactivity) compared to the Djungarian hamster. In this study, we compared locomotor activity of the hamsters in different environments, and compared their brain monoamine and metabolite levels to identify the mechanism by which both hamsters move differently. Activity of Roborovskii hamsters was significantly higher than Djungarian hamsters in the open field, while no difference was observed in their home cage. Dopamine (DA) and serotonin (5-HT) levels in the whole brain of Roborovskii hamster were significantly lower and their metabolic turnover rates were significantly higher than those of the Djungarian hamster. We conclude that the difference in activity under the novel environment between both species is partly, but not entirely, explained by the difference in monoamine levels and their metabolism in the brain. Key words: Djungarian hamster, locomotor activity, monoamine, open field, Roborovskii hamster Introduction Both Djungarian hamsters (Phodopus sungorus) and Roborovskii hamsters (Phodopus roborovskii) belong to the same genus of Phodopus. The Djungarian hamster has been widely used in chronobiological, thermophysiological or endocrinological research [18]. In contrast, the Roborovskii hamster, or Desert hamster, is rarely used as a laboratory animal. Djungarian and Roborovskii hamsters have substantial structural and behavioral differences [16]. In particular, we observed in preliminary experiments that Roborovskii hamsters show much more activity than Djungarian hamsters in the light-dark environment. Furthermore, the Roborovskii hamster was difficult to acclimate to a new environment. This may be attributable to the high locomotor activity of Roborovskii hamsters. This behavioral pattern of Roborovskii hamsters is similar to that of some hyperactive dysfunction like attention-deficit hyperactivity disorder (ADHD) or problematic behaviors of companion animals. ADHD is characterized by hyperactivity together with inattention and impulsivity [2, 15], and approximately 8 to 12% of children worldwide are believed to have ADHD [2]. Clinically, children with ADHD are treated (Received 25 February 2008 / Accepted 29 May 2008) Address corresponding: M. Furuse, Laboratory of Advanced Animal and Marine Bioresources, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka , Japan

2 448 Y. Kabuki, ET AL. with dopamine-based psychostimulants [5]. Nevertheless the neurobiological basis of ADHD remains poorly defined, although the effectiveness of these drugs suggests that dopamine neurotransmission is involved in the ADHD symptom in one way or another. In companion animals, hyperactivity in dogs is a common problematic behavior [9]. Hyperactive dogs have some features like tachycardia, panting, salivation, antidiuresis, lack of trainability, and failure to habituate to external stimulation. In most cases, symptoms of hyperactivity are due to environmental factors like lack of breed. However, some cases of hyperactivity are due to neurophysiological disorders. Since laboratory dogs that have the symptoms were initially identified as an animal model for ADHD, it was thought that the mechanism of hyperactivity in dogs is similar to ADHD. In the present study, we compared both Djungarian and Roborovskii hamsters to understand their behavioral characteristics and to explore the factors causing hyperactivity in the Roborovskii hamster as a potential animal model for hyperactive disorders including ADHD. We examined their activity in different environments, and measured monoamine levels of the whole brain to reveal the mechanism for hyperactivity. Materials and Methods Animals Male Djungarian hamsters (n=8) and Roborovskii hamsters (n=8), 3 weeks of age, reared under controlled environments were purchased from a local pet shop (Nomura, Fukuoka, Japan). The hamsters were housed individually in plastic cages ( cm) and allowed ad libitum access to a commercial diet (MF; Oriental Yeast, Tokyo, Japan) and water. A 12-h light/dark cycle was maintained throughout the experiment, with lights on at 08:00 and off at 20:00. Room temperature was maintained at 23 ± 1 C. After 10 day acclimation, the animals were subjected to two behavioral tests. The experimental procedures followed the guidance for Animal Experiments in the Faculty of Agriculture and in the Graduate School of Kyushu University, and Japanese Law (No. 105) and Notification (No. 6) by the Government. Behavioral assessments Spontaneous activity in the home cage Spontaneous activity was measured for 24 h in home cages. Activity was counted with an infrared beam sensor (NS-AS01; Neuroscience Inc., Tokyo, Japan) placed about 20 cm above the center of the cage, and analyzed using software, DAS-008 (Neuroscience Inc.). Open field test After the measurement of spontaneous activity in home cages, locomotor activity in a novel environment was recorded employing the open field test. This test was performed at 09:30 on the two days after the home cage activity test. Hamsters were individually transferred to the open field arena from the home cages. The arena was circular (diameter 60 cm and height 35 cm), and made of black takiflex. The field was covered with a white plastic sheet so that the sensor could recognize the dark color of the hamsters. The test started by placing the animal at the center of the arena. The behavior of animals was then observed for 5 min under dim light, 100 lux. After each test, the field was cleaned with an ethanol-water solution. The following behavioral categories were analyzed: distance of path, time the animals spent moving, movement speed of animals, and frequency of defecation. All behaviors except for defecation were analyzed automatically with a computer-based video tracking system (AXIS-90, Neuroscience, Inc). The frequency of defecation was manually recorded. Monoamine assessments All animals were decapitated immediately following completion of the open field test. Whole brains were immediately removed, weighed and kept at 80 C until analyzed. The levels (contents/mg wet tissue) of monoamines and their metabolites were determined using HPLC with electrochemical detection. Dopamine (DA), serotonin (5-HT), norepinephrine (NE) and epinephrine (E), and the DA metabolite dihydroxyphenylacetic acid (DOP- AC), 5-HT metabolite 5-hydroxyindoleacetic acid (5- HIAA), and NE and E metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured. Briefly, the tissue was homogenized and deproteinized in 0.2 M perchloric acid contain 100 µm EDTA 2Na. The homogenate was left for 30 min to deproteinize. Then, the homogenate

3 LOCOMOTOR ACTIVITY AND MONOAMINE IN HAMSTERS 449 Fig. 1. Spontaneous activity of Djungarian and Roborovskii hamsters over 24 h in their home cages. Values are mean ± SEM. was centrifuged at 10,000 g for 15 min at 0 C. After centrifugation, the supernatant was adjusted to ph 3 by adding 1 M CH 3 COOH. The supernatant was then centrifuged with a centrifuge-filtration unit (Ultra Free C3- GV Millipore, Bedford, MA, USA) at 10,000 g for 5 min at 0 C. Filtrate, 30 µl, was injected into an HPLC system (Eicom, Kyoto, Japan) with a mm ODS column (SC-5ODS, Eicom). The mobile phase, ph 3.5, consisted of 0.1 M aceto-citric acid buffer, 17% methanol, 190 mg/l sodium 1-octane sulfonate and 5 mg/l EDTA 2Na. Monoamines and their metabolites were detected using an electrochemical detector (ECD-300, Eicom, Kyoto, Japan) at an applied potential of V. Statistical analysis Home cage tests were analyzed with repeated measure ANOVA. Open field tests and monoamine assessments were analyzed with Student s t-test. Differences with P<0.05 were considered significant. Data were expressed as means ± SEM. Results Behavioral assessments Spontaneous activity in the home cage As shown in Fig. 1, no significant (F(1,13)=0.53, P>0.05) differences in spontaneous activity were observed between Djungarian and Roborovskii hamsters. The activity was significantly (F(23,299)=5.871, P<0.0001) different dependent on time, being higher in the dark period. No significant (F(23,299)=1.356, P>0.05) interaction between species and time was detected. Open fi eld test Fig. 2 shows the data for the open field test. The distance traveled by the Roborovskii hamster was significantly (P<0.0001) longer than that of the Djungarian hamster during the 5 min locomotor test (Fig. 2a). The Roborovskii hamster spent significantly (P<0.0001) more time moving than the Djungarian hamster (Fig. 2b). The movement speed of the Roborovskii hamster was significantly (P<0.0001) faster than that of the Djungarian hamster (Fig. 2c). No difference between the species was observed in frequency of defecation (data not shown). Monoamine assessments Table 1 shows the levels of monoamines and their metabolites. The levels of DA and 5-HT in the whole brain of the Roborovskii hamster were significantly (P<0.0005) lower than those of the Djungarian hamster. The DA metabolite, DOPAC, and 5-HT metabolite, 5-HIAA, levels in the whole brain of the Roborovskii

4 450 Y. KABUKI, ET AL. Fig. 2. (a) Distance of path, (b) moving time, and (c) movement speed of Djungarian and Roborovskii hamsters in the open field over 5 min. Values are mean ± SEM. *P< Table 1. Monoamines and their metabolites in the whole brain of Djungarian and Roborovskii hamsters DA*** DOPAC** 5-HT*** 5-HIAA** NE* E* MHPG Djungarian hamster 523 ± ± ± ± ± ± ± 5.7 Roborovskii hamster 424 ± ± ± ± ± ± ± 2.1 DA: Dopamine, DOPAC: dihydroxyphenyl acetic acid, 5-HT: serotonin, 5-HIAA: 5-hydroxyindoleacetic acid, NE: norepinephrine, E: epinephrine, MHPG: methoxyhydroxyphenylglycol. Values are the mean pg/mg wet tissue ± SEM. *P<0.05, **P<0.005, ***P<0.0005, significant difference between Djungarian hamster and Roborovskii hamster. Table 2. Monoamine metabolic turnover rates in the whole brain of Djungarian and Roborovskii hamsters DOPAC/DA*** 5-HIAA/5-HT*** MHPG/NE Djungarian hamster ± ± ± Roborovskii hamster ± ± ± DA: Dopamine, DOPAC: dihydroxyphenyl acetic acid, 5-HT: serotonin, 5-HIAA: 5-hydroxyindoleacetic acid, NE: norepinephrine, MHPG: methoxyhydroxyphenylglycol. Values are the mean ± SEM. *P<0.05, **P<0.005, ***P<0.0005, significant difference between Djungarian hamster and Roborovskii hamster. hamster were significantly (P<0.005) higher than those of the Djungarian hamster. The NE level in the whole brain of the Roborovskii hamster was significantly (P<0.05) lower, and the E level was significantly (P<0.05) higher than those of the Djungarian hamster. There were no significant differences in MHPG level in whole brains of the two species. Table 2 gives metabolic turnover rates of the monoamines. The metabolic turnover rates of DA (DOPAC/ DA) and 5-HT (5-HIAA/5-HT) in the Roborovskii hamster were significantly (P<0.0001) higher than those of the Djungarian hamster. There were no significant differences between the species in the metabolic turnover rate of NE (MHPG/NE). Discussion In the present study, we assessed activity in the home

5 LOCOMOTOR ACTIVITY AND MONOAMINE IN HAMSTERS 451 cage and open field. Monoamine levels in whole brains of Djungarian and Roborovskii hamsters were determined to elucidate the mechanism behind the difference in activity between the two hamsters. In the home cage, both species showed a similar pattern of spontaneous activity and nocturnal activity with no difference in activity observed between the two species. In the open field test, the Roborovskii hamster clearly moved more (hyperactive) than the Djungarian hamster with longer distance of path, and moving time, and higher speed. These findings indicate that the Roborovskii hamster is sensitive to a novel environment. ADHD has been studied using animal models such as the spontaneously hypertensive rat (SHR) [8], Naples high excitability (NHE) rat [17], and the DA transporter knock-out mouse [13]. In the present study, Roborovskii hamsters did not display hyperactivity in the home cage, whereas they displayed hyperactivity in a novel environment. This behavior is similar to that of NHE rats [17]. Therefore, the Roborovskii hamster is a potential animal model for hyperactive symptoms such as ADHD and high excitability symptom. However, the present study did not evaluate inattention and impulsivity associated with hyperactivity which will have to be addressed in future studies. Some dopaminergic stimulants like amphetamine and methylphenidate are clinically used in the therapy of ADHD children. ADHD model animals have several abnormalities in DA and NE neurotransmission. These facts indicate that DA dysfunction is involved in ADHD [2, 5, 14]. Furthermore, some reports suggest that 5-HT is also involved in hyperactivity [1]. In the present study, significant differences of monoamine levels between the Roborovskii and Djungarian hamsters were observed. Lower DA and 5-HT levels and higher turnover rates in the Roborovskii hamster may be associated with hyperactivity. Madras et al. [10] reported that a decrease in extrasynaptic DA concentration induced by increase of DA transporter was indicated in ADHD. In the same way, hyperactivity in the Roborovskii hamster may be caused by low extrasynaptic DA concentrations accompanied with low levels of DA in the whole brain. Elevated 5-HT concentration following DA depletion is suggested to cause hyperactivity [1]. In the present study, though, Djungarian hamsters showed lower activity and a higher level of 5-HT compared to Roborovskii hamsters. Furthermore, Roborovskii hamsters showed lower NE levels and higher E levels compared to Djungarian hamsters. There are inconsistent reports about the relationships between NE neurotransmission and hyperactivity. It was reported that depletion of NE release reduced the locomotor activity in the hyperactive mouse mutant coloboma [6], while the selective NE transporter inhibitor atomoxetine alleviated symptoms of ADHD [3]. NE neurotransmission is also involved in impulsive behavior, since enhancing NE neurotransmission decreased impulsive action [12]. Perhaps, low levels of NE in the Roborovskii hamster may be caused by impulsive action, and this action may lead to hyperactive behavior. The physiological function of E in the central nervous system was unclear in the present study. Monoamine levels are affected by enzymes including phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), and aromatic L- amino acid decarboxylase (AADC) [7]. These enzymes influence catecholamine and 5-HT levels in the brain [4]. In fact, different expressions of TH in some ADHD model animals were observed compared with normal animals [11]. The differences of monoamine levels in the whole brain between Djungarian and Roborovskii hamsters may be caused by differences in expression level or activity of these enzymes. In conclusion, we found differences in activity and monoamine levels in the whole brain between Djungarian and Roborovskii hamsters. Hence the difference in activity between both species may be explained in part by differences in monoamine levels and metabolism in the brain. Furthermore, Roborovskii hamsters may prove to be an animal model for hyperactivity symptom. However, in the present study, we only used male animals and sex differences in behaviors between the two hamster species should be examined in future. Acknowledgment(s) The authors are grateful to Dr. D.M. Denbow, Virginia Polytechnic Institute and State University, USA, for his reading of the manuscript.

6 452 Y. Kabuki, ET AL. References 1. Avale, M.E., Nemirovsky, S.I., Raisman-Vozari, R., and Rubinstein, M Elevated serotonin is involved in hyperactivity but not in the paradoxical effect of amphetamine in mice neonatally lesioned with 6-hydroxydopamine. J. Neurosci. Res. 78: Biederman, J. and Faraone, S.V Attention-deficit hyperactivity disorder. Lancet 366: Bymaster, F.P., Katner, J.S., Nelson, D.L., Hemrick-Luecke, S.K., Threlkeld, P.G., Heiligenstein, J.H., Morin, S.M., Gehlert, D.R., and Perry, K.W Atomoxetine increases extracellular levels of norepinephrine and dopamine in prefrontal cortex of rat: a potential mechanism for efficacy in attention deficit/hyperactivity disorder. Neuropsychopharmacology 27: Hyland, K Inherited disorders affecting dopamine and serotonin: critical neurotransmitters derived from aromatic amino acids. J. Nutr. 137: 1568S 1572S. 5. Iversen, S.D. and Iversen, L.L Dopamine: 50 years in perspective. Trends Neurosci. 30: Jones, M.D. and Hess, E.J Norepinephrine regulates locomotor hyperactivity in the mouse mutant coloboma. Pharmacol. Biochem. Behav. 75: Fernstrom, J.D. and Fernstrom, M.H Tyrosine, phenylalanine, and catecholamine synthesis and function in the brain. J. Nutr. 137: 1539S 1547S. 8. Li, Q., Lu, G., Antonio, G.E., Mak, Y.T., Rudd, J.A., Fan, M., and Yew, D.T The usefulness of the spontaneously hypertensive rat to model attention-deficit/hyperactivity disorder (ADHD) may be explained by the differential expression of dopamine-related genes in the brain. Neurochem. Int. 50: Luescher, U.A Hyperkinesis in dogs: six case reports. Can. Vet. J. 34: Madras, B.K., Miller, G.M., and Fischman, A.J The dopamine transporter and attention-deficit/hyperactivity disorder. Biol. Psychiatry 57: Oades, R.D., Sadile, A.G., Sagvolden, T., Viggiano, D., Zuddas, A., Devoto, P., Aase, H., Johansen, E.B., Ruocco, L.A., and Russell, V.A The control of responsiveness in ADHD by catecholamines: evidence for dopaminergic, noradrenergic and interactive roles. Dev. Sci. 8: Pattij, T. and Vanderschuren, L.J The neuropharmacology of impulsive behaviour. Trends pharmacol. Sci. 29: Seeman, P., Hall, F.S., and Uhl, G Increased dopamine D2 high receptors in knockouts of the dopamine transporter and the vesicular monoamine transporter may contribute to spontaneous hyperactivity and dopamine supersensitivity. Synapse 61: Solanto, M.V Dopamine dysfunction in AD/HD: integrating clinical and basic neuroscience research. Behav. Brain Res. 30: Spencer, T.J., Biederman, J., and Mick, E Attentiondeficit/hyperactivity disorder: diagnosis, lifespan, comorbidities, and neurobiology. J. Pediatr Psychol. 32: Spyropoulos, B., Ross, P.D., Moens, P.B., and Cameron, D.M The synaptonemal complex karyotypes of palearctic hamsters, Phodopus roborovskii Satunin and P. sungorus Pallas. Chromosoma 86: Viggiano, D., Vallone, D., and Sadile, A Dysfunctions in dopamine systems and ADHD: evidence from animals and modeling. Neural Plast. 11: Weiner, J. and Heldmaier, G Metabolism and thermoregulation in two races of Djungarian hamsters: Phodopus sungorus sungorus and P. s. campbelli. Comp. Biochem. Physiol., A 86: