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Behavioral lateralization in the T-maze and monoaminergic brain asymmetries
Physiology & Behavior, Vol. 40, pp. 785--789. Copyright © Pergamon Journals Ltd., 1987. Printed in the U.S.A.
0031-9384/87 $3.00 + .00
BRIEF COMMUNICATION
Behavioral Lateralization in the T-Maze and Monoaminergic Brain Asymmetries M. D. D I A Z P A L A R E A , M. C. G O N Z A L E Z
A N D M. R O D R I G U E Z
Department o f Physiology, School o f Medicine, La Laguna University Tenerife, Canary Islands, Spain R e c e i v e d 16 D e c e m b e r 1986 DIAZ PALAREA, M. D., M. C. GONZALEZ AND M. RODRIGUEZ. Behavioral lateralization in the T-maze and monoaminergic brain asymmetries. PHYSIOL BEHAV 40(6) 785-789, 1987.--Recently we have reported a marked rat lateralization in the T-maze choice. The present study examines the relationship between the ascending monoaminergic systems and the T-maze behavioral asymmetry. There were no significant differences for serotonin or norepinepln-ine between the T-maze preferred and non-preferred brain sides in the s. nigra, ventral tegmental area, striatum, acumbens, frontal lobe or hippocampus. Only in the hippocampus was dopamine concentration significantly greater for the brain site ipsilateral to the T-maze choice side. Previously, we reported that both apomorphine, a dopamine receptor agonist, and 6-hydroxydopamine lesion in the medial forebraln bundle of the catecholaminerglc neurons affect the T-maze asymmetry; we therefore suggested that the T-maze choice could be related with the ascending dopaminerglc systems. The present data strongly support this hypothesis and suggest that the DA cells involved in the spatial asymmetry in the T-maze are included in the dopaminergic mesohippocampal system. Brain asymmetry
Dopamine
Norepinephrine
Serotonin
T-maze
monoaminergic synaptic asymmetry are striatum [14, 15, 27], frontal lobe [28], n. accumbens [24] and the hippocampus [12]. In the present study we measured the dopamine, norepinephrine and serotonin levels in these brain areas, as well as in the s. nigra and ventral tegmental area of both brain sides.
F O R many years it was thought that humans were unique in having functional brain lateralization at population levels. More recently, there have been various studies on animals reporting functional asymmetries with population right-bias [7-9, 17, 25]. However, the population lateralization demonstrated for animals is not of the magnitude seen in the human (between 54--59%). Recently, we reported a marked rat population lateralization in the T-maze choice (68.57% of right-biased; 17.94% of the left-biased and 14.28% nonbiased rats) [6]. This important asymmetry in rats' behavior support the hypothesis that phylogenic selection pressures is the cause of brain asymmetry. There is increasing evidence demonstrating chemical asymmetries in the brain. Asymmetries have been described in dopamine content [14,32], dopamine receptors [27], dopamine metabolism [31] and dopamine-stimulated adenyl cyclase activity [18]. Previously, we have reported that both the dopamine receptor agonist apomorphine and the 6hydroxydopamine lesion of the ascending catecholaminergic system modify the behavioral asymmetry in the T-maze [6]. This previous study suggests that catecholaminergic systems, and especially the DA-ascending neurons, could be involved in the T-maze behavioral lateralization. The present study examined the relationship between the ascending monoaminergic systems and the T-maze behavioral asymmetry. The brain structures previously related to
METHOD
Subjects Experiments were c a r d e d out on male Sprague-Dawley rats (Panlab, Barcelona) weighing 200-250 g. Twelve animals were housed under normal laboratory conditions of 22-+ I°C on a standard light-dark schedule (12:12 lights 300-1500 hr and given free access to standard laboratory diet and water, except during the behavioral measurement).
Behavioral Testing The rats were tested for spatial preference [6,32] in an electrified glass-wall T-maze. The stem was 30 cm long, the arms were each 25 cm long and the walls 8 cm high. The floor o f the stem was a grid o f 0.4 cm stainless steel rods, spaced 1.3 cm apart. The maze was centrally placed in a sound attenuating room and both arms were cleaned regularly to eliminate odors. The behavioral tests were performed be-
785
786
DIAZ P A L A R E A , G O N Z A L E Z AND RODRIGUEZ
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FIG. 2. Absolute % of laterality in the T-maze for 11 succesive days. ANOVA analysis revealed significant differences (F=4.96, p<0.0001). The paired t-test shows: (1) Day 1 vs. Day 3, t=-2.60, p=0.024; (2)Day 1 vs. Day 4, t=-2.60,p=0.024; (3)Day 1 vs. Day 5, t=-4.00, p=0.0021; (4) Day 1 vs. Day 6, t =-4.06,p =0.0019; (5) Day 1 vs. Day 7, t = -5.00, p =0.0004; (6) Day 1 vs. Day 8, t = -4.84, p =0.005; (7) Day 1 vs. Day 9, t =-4.17, p =0.0016; (8) Day 1 vs. Day 10, t=-5.00, p=0.001M; (9)Day 1 vs. Day 11, t=-8.86, p<0.0001. Values represent mean (_+S.E.M.) values of% laterality.
FIG. 1. Localization of the dissected tissue pieces. Slice 1: from optic chiasm to 1 mm anterior to chiasm. Slice 2: from optic chiasm to 1 mm posterior to optic chiasm. Slice 3: from 3 mm posterior chiasm to 4.5 mm posterior to optic chiasm. Slice 4: from 0.2 mm anterior to the ventral anterior pontine fibres to 1.2 mm anterior of pontine fibres. The frontal lobe (FL) and the n. accumbens (Ac) was dissected from slice 1; striatum (CP) from slice 2; hippocampus (H) from slice 3; s. nigra (SN) and ventral tegmental area (VTA) from slice 4. tween 5-9 hr of the light illumination. Rats were placed in the stem of the T-maze and a scrambled 0.4 mA current was applied to the grid by a Letica LI-100=20 shocker. The shock was terminated when a rat entered either the left or right arm of the T-maze. The rat was then removed from the arm and returned to the stem. Testing consisted of five consecutive trials. Each rat was tested during 12 successive days. The percent of laterality for each rat and day is the absolute value of the formula:
schematically illustrated in Fig. 1. The brain areas were immediately cooled by liquid nitrogen and stored at -79°C until analyzed. Pieces of right and left striatum, frontal lobe, accumbens, hippocampus, ventral tegmental area and s. nigra (Fig. 1) were weighed in conical 1.5 ml test tubes and 300/xl 0.1 M perchloric acid containing 4×10 -~ M sodium metabisulphite, was pipetted into the tubes. The mixture was sonicated at 100 W for about 12 sec while on ice, and the homogenate was centrifuged for 15 min at 15000×g. An aliquot of the supernatant was injected into the chromatographic column (300×3.9 mm stainless steel column packed with/xBondapak C18, 19/zm, Waters Assoc., Milford, MA. The mobile phase consisted of 0.07 M N a H2PO4.H20, 0.1 mM EDTA:methanol (90:10, v/v), containing hexyl sulfate 1.7x 10-3 M. The final solution (pH=3.4) was filtered (0.45 /xm Millipore f'dter) before use. Standard DA, N A and 5-HT (Sigma, St. Louis) were dissolved in 0.1 M perchloric acid containing 4 x 1 0 -5 M sodium metabisulphite and kept as stock solutions at -20°C. Quantitations were performed from standard curves of peak height. The standard curves were reproducible from day to day, thus further simplifying' the quantitation procedure. Concentration of monoamines in tissue samples were expressed as ng/mg protein. Statistical Analysis
right-side - left-side
x 100
right-side + left-side Biochemical Analyses In this paper we use a procedure that permits the simultaneous determination of dopamine (DA), norepinephrine (NA) and serotonin (5-HT) in a sample. This procedure uses high performance liquid chromatography combined with electrochemical detection as described to Magnusson et al. [23] and Gonzalez et al. [19]. Rats were sacrificed by decapitation and the brains quickly removed and dissected on ice. The regional dissection of the brain was c a r d e d out as
Biochemical and behavioral data were statistically evaluated using a one-way analysis of variance (ANOVA) followed by Student's t-test for related samples. Differences were judged significant when associated with a probability of 5% or less. RESULTS The percentages of laterality in the T-maze test is shown in Fig. 2. As in the previous study [6] the data reveal a progressive increase of laterality in the succesives days [ANOVA with F(10)=4.96, p<0.0001]. Rats were separated in right- or left-preference groups according to their T-maze response over the previous 6 test days. 83.34% of the rats
BEHAVIORAL
LATERALIZATION
787 TABLE 1
DOPAMINE, NOREPINEPHRINE AND SEROTONIN LEVELS IN THE RIGHT AND LEFT BRAIN SIDE Dopamine Right Side S. Nigra VTA Striatum Accumbens Frontal L Hippocampus
9.4 15.8 35.4 68.3 2.2 1.3
Norepinephrine Left Side
_+ 0.9 _+ 1.9 _+ 12.4 _+ 9.2 _+ 0.6 -+ 0.3
9.8 15.3 32.9 74.6 2.3 0.9
+ 0.8 -+ 2.6 -+ 10.4 __ 10.1 _+ 0.9 -+ 0.3
Right Side 8.2 17.9 7.8 22.7 8.5 6.6
-+ 1.4 _+ 2.2 -+ 1.6 __ 7.6 -+ 1.3 -+ 0.7
Left Side 6.9 23.0 8.3 18.1 8.9 6.8
-+ 0.7 -+ 4.4 -- 1.6 -+ 7.4 -+ 1.1 -+ 1.0
Serotonin Right Side 24.8 26.3 15.8 26.0 5.4 7.4
_+ 3.0 _+ 3.2 _+ 1.8 _+ 2.0 -+ 0.7 -+ 0.6
Left Side 22.6 26.7 16.4 23.9 5.5 7.8
-+ 3.1 _+ 3.5 -+ 2.4 - 2.1 _+ 0.6 _+ 0.7
Values are the mean (-+S.E.M.) of monoamine concentration (ng/mg protein). *p=0.015 ipsi- vs. cant differences between right and left catecholamines concentration in the brain areas studied. The paired t-test shows: DA striatum, t =0.38, p =0.7148; DA s. nigra, t = -0.36, p =0.7237; DA n. accumbens, t = - l . 1 4 , p=0.2777; DA VTA, t=0.22, p=0.8327; DA frontal lobe, t = - 0 . 0 7 , p=0.9492; DA hippocampus, t=0.98, p=0.3490; NA striatum, t = - 0 . 2 1 , p=0.8421; NA s. nigra, t = 1.29, p=0.2335; N A n . accumbens, t = l . 2 0 , p=0.2570; NA VTA, t = - 1 . 4 2 , p=0.1930; NA frontal lobe, t = - 0 . 4 5 , p =0.6609; NA hippocampus, t = - 0 . 1 5 , p =0.8856; 5-HT striatum, t = - 0 . 2 1 , p =0.8399; 5-HT s. nigra, t=0.62, p=0.5524; 5-HT n. accumbens, t=0.89, p=0.3936; 5-HT VTA, t = - 0 . 2 1 , p=0.8369; 5-HT frontal lobe, t = - 0 . 1 5 , p =0.8862; 5-HT hippocampus, t = - 0 . 6 4 , p =0.5372.
TABLE 2 DOPAMINE, NOREPINEPHRINE AND SEROTONIN LEVELS IN THE IPSI- AND CONTRA-LATERAL BRAIN SIDE RELATED TO THE PREFERRED DIRECTION IN THE T-MAZE TEST Dopamine IpsiLateral S. Nigra VTA Striatum Accumbens Frontal L Hippocampus
9.2 15.4 36.0 69.0 2.2 1.5
_+ 0.9 _+ 1.9 _+ 12.2 _+ 8.8 -+ 0.6 _+ 0.4
Norepinephrine
ContraLateral 9.9 15.6 32.3 73.8 2.2 0.6
-+ 0.9 -4- 2.6 -+ 10.6 -+ 10.5 _+ 0.9 -+ 0.2
IpsiLateral 8.0 17.7 7.2 22.7 8.5 6.6
-+ 0.4 -+ 2.2 _+ 1.6 _+ 7.6 -+ 1.3 _+ 0.4
ContraLateral 7.0 23.1 8.8 18.1 8.8 6.7
-+ 0.7 --- 4.3 -+ 1.6 -+ 4.1 -+ 1.1 -+ 0.5
Serotonin IpsiLateral 24.7 26.3 15.8 25.9 5.4 7.1
_+ 3.0 _+ 3.2 -+ 1.8 -+ 2.0 _+ 0.7 -+ 0.5
ContraLateral 22.6 26.7 16.4 23.9 5.4 8.0
-+ 3.1 _+ 3.5 _+ 2.4 -+ 2.1 _+ 0.6 -+ 0.7
Values are the mean (-+S.E.M.) of monoamine concentration (ng/mg protein). *p=0.016 ipsi- vs. contra-lateral side, t(l 1)=2.85, for the hippocampus. There were no significant differences for the other brain areas studied. The paired t-test shows: DA striatum, t=0.56, p=0.5892; DA s. nigra, t = - 0 . 6 3 , p=0.5435; D A n . accumbens, t = - 0 . 8 5 , p=0.4145; DA VTA, t = - 0 . 0 9 , p=0.9292; DA frontal lobe, t=-0.04,p =0.9672; NA striatum, t = - 0 . 7 5 , p = 0 . 4 7 4 2 ; NA s. nigra, t =0.99,p=0.3516; N A n . accumbens, t = 1.19, p =0.2601; NA VTA, t = - 1.55, p=0.1594; NA frontal lobe, t = - 0 . 2 9 , p =0.7765; NA hippocampus, t=-O. 19, p=0.8527; 5-HT striatum, t = - 0 . 2 2 , p=0.8397; 5-HT s. nigra, t=0.63, p=0.552; 5-HT n. accumbens, t=0.88, p=0.396; 5-HT VTA, t = - 0 . 2 3 , p=0.8362; 5-HT frontal lobe, t = -0.13, p =0.88; 5-HT hippocampus, t = - 1.41, p =0.1862.
s h o w f i g h t - p r e f e r e n c e . T h e s e r e s u l t s are c o n s i s t e n t w i t h t h e p o p u l a t i o n f i g h t - b i a s e d l a t e r a l i z a t i o n in t h e T - m a z e t h a t w e h a v e r e c e n t l y r e p o r t e d [6]. T h e r e s u l t s o f b i o c h e m i c a l a n a l y s i s in the s t r i a t u m , acc u m b e n s , f r o n t a l lobe, s. nigra, v e n t r a l t e g m e n t a l a r e a a n d h i p p o c a m p u s a p p e a r s in T a b l e 1 (for fight a n d left b r a i n side) a n d T a b l e 2 (for t h e T - m a z e p r e f e r r e d direction). T h e s e d a t a d o n o t d e m o n s t r a t e a significant d i f f e r e n c e in n o r e p i n e p r h i n e o r s e r o t o n i n l e v e l s b e t w e e n v a l u e s o f b r a i n sites c o n t r a l a t e r a / t o t h e T - m a z e p r e f e r r e d d i r e c t i o n c o m p a r e d to v a l u e s for t h e ipsilateral b r a i n sites. D o p a m i n e v a l u e s w e r e t h e s a m e for t h e b r a i n a r e a s s t u d i e d e x c e p t f o r t h e h i p p o c a m p u s , f o r w h i c h a m a r k e d a s y m m e t r y w a s f o u n d (Table 2). T h e
d o p a m i n e c o n c e n t r a t i o n w a s significantly h i g h e r for t h e b r a i n side ipsilateral to t h e T - m a z e c h o i c e , t ( 1 1 ) = 2 . 8 5 , p = 0 . 0 1 5 9 . T h e r e are n o statistical d i f f e r e n c e s b e t w e e n t h e right a n d left side in the b r a i n a r e a s s t u d i e d (Table 1). DISCUSSION T h e d e m o n s t r a t i o n o f f u n c t i o n a l a s y m m e t r i e s in a n i m a l s a p p e a r s to s u p p o r t t h e h y p o t h e s i s t h a t biologically determined asymmetries have persisted throughout vertebrate e v o l u t i o n , a n d t h a t h u m a n a s y m m e t r i e s are o n l y a n e x a m p l e of a fundamental feature of vertebrate neuroanatomy and b e h a v i o r [13]. P r e v i o u s l y , we h a v e r e p o r t e d a m a r k e d
788
DIAZ PALAREA, G O N Z A L E Z AND RODRIGUEZ
right-biased lateralization in the T-maze test at both individual and population levels (85.7% of the rats presented a behavioral asymmetry, 80% being right-biased within the lateralized group). In the present study, we report that spatial asymmetry in the T-maze is related to an imbalance between left and right DA levels of the hippocampus. Initially, a histochemical study suggested that the presence of dopamine in the hippocampus is independent of noradrenergic neurons [20]. However, other authors assumed that hippocampal DA only serves as the precursor to norepinephrine [5,22]. Evidence for a transmitter role of dopamine in the rat hippocampal formation has been provided more recently [3, 21, 26]. Thus, several studies reported the existence in the hippocampus of both a DA-sensitive receptor site [2] and DA-sensitive adenylate cyclase [12]. Moreover, haloperidol and apomorphine induce biochemical modification in the hippocampus similar to that observed in other DA-rich brain areas. The 6hydroxydopamine-induced destruction of ascending noradrenergic pathways failed to affect hippocampal DOPAC levels and only slightly reduced DA content in comparison with the almost total depletion of norepinephrine [3]. In conclusion, the previous biochemical findings suggest the existence of DA-synapses in the hippocampal formation. This hypothesis is also supported by the previous study of
Ishikawa et al. [21] who reported differences in the intrahippocampal distribution of norepinephrine and dopamine. The present experiment revealed that the interhemispheric balance of catecholamines in the hippocampus is different for dopamine and norepinephr~me, and therefore is a new evidence that hippocampal DA is independent of the noradrenergic system. The hippocampus is a brain structure previously related to various functions including spatial organization of behavior [1,4]. In spite of the reported morphological [11,10], biochemical [30] and functional [29] asymmetries in the hippocampus of nonhuman species, the possible role of DA-innervation has not been previously evaluated. In the present study we report a new biochemical asymmetry for the hippocampus related to dopamine innervation. In addition, our data strongly suggest that this asymmetric distribution of dopamine is related to the spatial organization of behavior in the T-maze test. As shown in Table 2, the DA levels in the homolateral hippocampus to the T-maze spatial preference is greater than in the contralateral side. In the present study, the NA/DA and 5-HT/DA ratios are similar to those previously reported by Ishikawa et al. [24]. The NA or 5-HT determinations reveal an interhemispheric balance in the brain areas studied, including the hippocampus. Therefore, we conclude that in the hippocampus, DA levels and not NA or 5-HT levels are related to spatial preference.
REFERENCES 1. Becker, J. T., J. A. Walker and D. S. Olton. Neuroanatomical bases of spatial memory. Brain Res 200: 307-320, 1980. 2. Bishoff, S., B. Scatton and J. Kork. Dopamine metabolism, spiperone binding and adenylate cyclase activity in the adult rat hippocampus after ingrowth of dopaminergic neurons from embryonic implants. Brain Res 179: 77-84, 1979. 3. Bishoff, S., B. Scatton and J. Off. Biochemical evidence for a transmitter role of dopamine in the rat hippocampus. Brain Res 165: 161-165, 1979. 4. Biozovski, D. E1 hipocampo y el comportamiento. Mun Ci 58: 516-523, 1986. 5. Brownstein, M., J. M. Saavedra and M. Palkovits. Norepinephrine and dopamine in the limbic system of the rat. Brain Res 79: 431-436, 1974. 6. CasteUano, M. D., M. D. Diaz-Palarea, M. Rodriguez and J. Barroso. Lateralization in male rats and dopaminergic system: Evidence of right-side population bias. Physiol Behav 40: 607-612, 1987. 7. Cowey, A. and T. Bozek. Contralateral neglect after unilateral dorsomedial prefrontal lesions in rats. Brain Res 72: 53-63, 1974. 8. Denenberg, W. N. and H. Fowler. Spontaneous alternation behaviour. Psychol Bull 55: 412-428, 1958. 9. Denenberg, V. H. Effects of right hemisphere lesions in rats. In: The Right Hemisphere. Neurology and Neuropsychology, edited by A. Ardila and F. Ostrosky-Solis. New York: Gordon and Breach, 1984, pp. 241-262. 10. Diamond, M. C., G. M. Kurphy and K. Akiama. Morphologic hippocampal asymmetry in male and female rats. Exp Neurol 76: 553-565, 1982. 11. Diamond, M. C. Age, sex, and environmental influences. In: Cerebral Dominance: The Biological Foundations, Chapter 9, edited by N. Geschwind, A. M. Galaburda. Cambridge, MA: Harvard University Press, 1984, pp. 134-146. 12. Dolphin, A. and J. Bockaert./3-Adrenergic receptors coupled to adenylate cyclase in cat brain: regional destribution, pharmacological characteristics and adaptative responsiveness. In: Recent Advances in the Pharmacology o f Adrenoceptors, edited by E. Szabadi et al. Amsterdam: Elsevier/North-
Holland, 1981.
13. Geschwind, N. and M. A. Gataburda. In: Cerebral Dominance: The Biological Foundations, edited by N. Geschwind and M. A. Galaburda. Cambridge, MA: Harvard University Press, 1984. 14. Glick, S. D., T. P. Jerussi, D. H. Waters and J. P. Green. Amphetamine-induced changes in striatal dopamine and acetylcholine levels and relationship to rotation (circling behavior) in rats. Biochem Pharmacol 23: 3223-3225, 1974. 15. Glick, S. D., R. D. Cox and S. Greenstein. Relationship of rats' spatial preferences to effects of d-amphetamine on timing behavior. Eur J Pharmacol 33: 173-182, 1975. 16. Glick, S. D., R. C. Meibach, R. D. Cox and S. Maayani. Phencyclidine-induced rotation and hippocampal modulation of nigrostriatal asymmetry. Brain Res 196: 9%107, 1980. 17. Glick, S. D. and D. A. Ross. Right-sided population bias and lateralization of activity in normal rats. Brain Res 205: 222-225, 1981. 18. Glick, S. D., R. C. Meibach, R. D. Cox and S. Maayani. Multiple and interrelated functional asymmetries in rat brain. Life Sci 32: 2215-2221, 1983. 19. Gonzalez, M. C., R. Arevalo, R. Castro, M. D. Diaz-Palarea and M. Rodriguez. Different roles of incertohypothalamic and nigrostriatal systems in the thermoregulatory responses of the rat. Life Sci 39: 707-715, 1986. 20. Hokfelt, T., A. Ljungdahl, K. Fuxe and O. Johansson. Dopamine nerve terminals in the rat iimbic cortex: aspects of the dopamine hypothesis of schizophrenia. Science 184: 177179, 1974. 21. Ishikawa, K., T. Ott and J. McGangh. Evidence for dopamine as a transmitter in dorsal hippocampus. Brain Res 232: 222-226, 1982. 22. Lindvall, O., A. Bjorklund, R. Moore and U. Stenevi. Mesencephalic dopamine neurons projecting to neocortex. Brain Res 81: 325-331, 1974. 23. Magnusson, O., L. B. Nilsson and D. Westerlund. Simultaneous determination of dopamine, DOPAC and homovanillic acid. Direct injection of supernatants from brain tissue homogenates in a liquid chromatography electrochemical detection system. J Chromatogr 221: 237-247, 1980.
BEHAVIORAL LATERALIZATION 24. Rosen, G. D., S. Finklestein, A. L. Stoll, D. A. Yutzey and V. H. Denenberg. Neurochemical asymmetries in the albino rat's cortex, striatum, and nucleus accumbens. Life Sci 34: 11431148, 1984. 25. Ross, D. A. and S. D. Glick. Lateralized effects of bilateral frontal cortex. Brain Res 210: 379-382, 1981. 26. Scatton, B., H. Simon, M. Moal and S. Bischoff. Orion of dopaminergic innervation of the rat hippocampal formation. Neurosci Lett 18: 125-131, 1980. 27. Schneider, L. H., R. B. Murphy and E. E. Coons. Lateralization of striatal dopamine (D2) receptors in normal rats. Neurosci Lett 33: 281-284, 1982. 28. Slopsema, J. S., J. Gugten and P. C. Bruin. Regional concentration of noradrenaline and dopamine in the frontal cortex of the rat: dopaminergic innervation of the prefrontal subareas and lateralization of prefrontal dopamine. Brain Res 250: 197-200, 1982.
789 29. Stokes, K. A. and D. C. McIntyre. Lateralized state-dependent learning produced by hippocampal kindled convulsions: Effect of split-brain. Physiol Behav 34: 217-224, 1985. 30. Valdes, J. J., C. Mactutus and R. N. Cory. Lateralization of norepinephrine, serotonin and choline uptake into hippocampal synaptosomes of sinistral rats. PhysiolBehav 27: 381-383, 1981. 31. Yamamoto, B. and C. R. Freed. Asymmetric dopamine and serotonine metabolism in nigrostriatal and limbic structures of trained circling rat. Brain Res 287: 115-119, 1984. 32. Zimmenberg, B., S. D. Glick and T. P. Jerussi. Neurochemical correlate of spatial preference in rats. Science 185: 623-625, 1974.
0031-9384/87 $3.00 + .00
BRIEF COMMUNICATION
Behavioral Lateralization in the T-Maze and Monoaminergic Brain Asymmetries M. D. D I A Z P A L A R E A , M. C. G O N Z A L E Z
A N D M. R O D R I G U E Z
Department o f Physiology, School o f Medicine, La Laguna University Tenerife, Canary Islands, Spain R e c e i v e d 16 D e c e m b e r 1986 DIAZ PALAREA, M. D., M. C. GONZALEZ AND M. RODRIGUEZ. Behavioral lateralization in the T-maze and monoaminergic brain asymmetries. PHYSIOL BEHAV 40(6) 785-789, 1987.--Recently we have reported a marked rat lateralization in the T-maze choice. The present study examines the relationship between the ascending monoaminergic systems and the T-maze behavioral asymmetry. There were no significant differences for serotonin or norepinepln-ine between the T-maze preferred and non-preferred brain sides in the s. nigra, ventral tegmental area, striatum, acumbens, frontal lobe or hippocampus. Only in the hippocampus was dopamine concentration significantly greater for the brain site ipsilateral to the T-maze choice side. Previously, we reported that both apomorphine, a dopamine receptor agonist, and 6-hydroxydopamine lesion in the medial forebraln bundle of the catecholaminerglc neurons affect the T-maze asymmetry; we therefore suggested that the T-maze choice could be related with the ascending dopaminerglc systems. The present data strongly support this hypothesis and suggest that the DA cells involved in the spatial asymmetry in the T-maze are included in the dopaminergic mesohippocampal system. Brain asymmetry
Dopamine
Norepinephrine
Serotonin
T-maze
monoaminergic synaptic asymmetry are striatum [14, 15, 27], frontal lobe [28], n. accumbens [24] and the hippocampus [12]. In the present study we measured the dopamine, norepinephrine and serotonin levels in these brain areas, as well as in the s. nigra and ventral tegmental area of both brain sides.
F O R many years it was thought that humans were unique in having functional brain lateralization at population levels. More recently, there have been various studies on animals reporting functional asymmetries with population right-bias [7-9, 17, 25]. However, the population lateralization demonstrated for animals is not of the magnitude seen in the human (between 54--59%). Recently, we reported a marked rat population lateralization in the T-maze choice (68.57% of right-biased; 17.94% of the left-biased and 14.28% nonbiased rats) [6]. This important asymmetry in rats' behavior support the hypothesis that phylogenic selection pressures is the cause of brain asymmetry. There is increasing evidence demonstrating chemical asymmetries in the brain. Asymmetries have been described in dopamine content [14,32], dopamine receptors [27], dopamine metabolism [31] and dopamine-stimulated adenyl cyclase activity [18]. Previously, we have reported that both the dopamine receptor agonist apomorphine and the 6hydroxydopamine lesion of the ascending catecholaminergic system modify the behavioral asymmetry in the T-maze [6]. This previous study suggests that catecholaminergic systems, and especially the DA-ascending neurons, could be involved in the T-maze behavioral lateralization. The present study examined the relationship between the ascending monoaminergic systems and the T-maze behavioral asymmetry. The brain structures previously related to
METHOD
Subjects Experiments were c a r d e d out on male Sprague-Dawley rats (Panlab, Barcelona) weighing 200-250 g. Twelve animals were housed under normal laboratory conditions of 22-+ I°C on a standard light-dark schedule (12:12 lights 300-1500 hr and given free access to standard laboratory diet and water, except during the behavioral measurement).
Behavioral Testing The rats were tested for spatial preference [6,32] in an electrified glass-wall T-maze. The stem was 30 cm long, the arms were each 25 cm long and the walls 8 cm high. The floor o f the stem was a grid o f 0.4 cm stainless steel rods, spaced 1.3 cm apart. The maze was centrally placed in a sound attenuating room and both arms were cleaned regularly to eliminate odors. The behavioral tests were performed be-
785
786
DIAZ P A L A R E A , G O N Z A L E Z AND RODRIGUEZ
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FIG. 2. Absolute % of laterality in the T-maze for 11 succesive days. ANOVA analysis revealed significant differences (F=4.96, p<0.0001). The paired t-test shows: (1) Day 1 vs. Day 3, t=-2.60, p=0.024; (2)Day 1 vs. Day 4, t=-2.60,p=0.024; (3)Day 1 vs. Day 5, t=-4.00, p=0.0021; (4) Day 1 vs. Day 6, t =-4.06,p =0.0019; (5) Day 1 vs. Day 7, t = -5.00, p =0.0004; (6) Day 1 vs. Day 8, t = -4.84, p =0.005; (7) Day 1 vs. Day 9, t =-4.17, p =0.0016; (8) Day 1 vs. Day 10, t=-5.00, p=0.001M; (9)Day 1 vs. Day 11, t=-8.86, p<0.0001. Values represent mean (_+S.E.M.) values of% laterality.
FIG. 1. Localization of the dissected tissue pieces. Slice 1: from optic chiasm to 1 mm anterior to chiasm. Slice 2: from optic chiasm to 1 mm posterior to optic chiasm. Slice 3: from 3 mm posterior chiasm to 4.5 mm posterior to optic chiasm. Slice 4: from 0.2 mm anterior to the ventral anterior pontine fibres to 1.2 mm anterior of pontine fibres. The frontal lobe (FL) and the n. accumbens (Ac) was dissected from slice 1; striatum (CP) from slice 2; hippocampus (H) from slice 3; s. nigra (SN) and ventral tegmental area (VTA) from slice 4. tween 5-9 hr of the light illumination. Rats were placed in the stem of the T-maze and a scrambled 0.4 mA current was applied to the grid by a Letica LI-100=20 shocker. The shock was terminated when a rat entered either the left or right arm of the T-maze. The rat was then removed from the arm and returned to the stem. Testing consisted of five consecutive trials. Each rat was tested during 12 successive days. The percent of laterality for each rat and day is the absolute value of the formula:
schematically illustrated in Fig. 1. The brain areas were immediately cooled by liquid nitrogen and stored at -79°C until analyzed. Pieces of right and left striatum, frontal lobe, accumbens, hippocampus, ventral tegmental area and s. nigra (Fig. 1) were weighed in conical 1.5 ml test tubes and 300/xl 0.1 M perchloric acid containing 4×10 -~ M sodium metabisulphite, was pipetted into the tubes. The mixture was sonicated at 100 W for about 12 sec while on ice, and the homogenate was centrifuged for 15 min at 15000×g. An aliquot of the supernatant was injected into the chromatographic column (300×3.9 mm stainless steel column packed with/xBondapak C18, 19/zm, Waters Assoc., Milford, MA. The mobile phase consisted of 0.07 M N a H2PO4.H20, 0.1 mM EDTA:methanol (90:10, v/v), containing hexyl sulfate 1.7x 10-3 M. The final solution (pH=3.4) was filtered (0.45 /xm Millipore f'dter) before use. Standard DA, N A and 5-HT (Sigma, St. Louis) were dissolved in 0.1 M perchloric acid containing 4 x 1 0 -5 M sodium metabisulphite and kept as stock solutions at -20°C. Quantitations were performed from standard curves of peak height. The standard curves were reproducible from day to day, thus further simplifying' the quantitation procedure. Concentration of monoamines in tissue samples were expressed as ng/mg protein. Statistical Analysis
right-side - left-side
x 100
right-side + left-side Biochemical Analyses In this paper we use a procedure that permits the simultaneous determination of dopamine (DA), norepinephrine (NA) and serotonin (5-HT) in a sample. This procedure uses high performance liquid chromatography combined with electrochemical detection as described to Magnusson et al. [23] and Gonzalez et al. [19]. Rats were sacrificed by decapitation and the brains quickly removed and dissected on ice. The regional dissection of the brain was c a r d e d out as
Biochemical and behavioral data were statistically evaluated using a one-way analysis of variance (ANOVA) followed by Student's t-test for related samples. Differences were judged significant when associated with a probability of 5% or less. RESULTS The percentages of laterality in the T-maze test is shown in Fig. 2. As in the previous study [6] the data reveal a progressive increase of laterality in the succesives days [ANOVA with F(10)=4.96, p<0.0001]. Rats were separated in right- or left-preference groups according to their T-maze response over the previous 6 test days. 83.34% of the rats
BEHAVIORAL
LATERALIZATION
787 TABLE 1
DOPAMINE, NOREPINEPHRINE AND SEROTONIN LEVELS IN THE RIGHT AND LEFT BRAIN SIDE Dopamine Right Side S. Nigra VTA Striatum Accumbens Frontal L Hippocampus
9.4 15.8 35.4 68.3 2.2 1.3
Norepinephrine Left Side
_+ 0.9 _+ 1.9 _+ 12.4 _+ 9.2 _+ 0.6 -+ 0.3
9.8 15.3 32.9 74.6 2.3 0.9
+ 0.8 -+ 2.6 -+ 10.4 __ 10.1 _+ 0.9 -+ 0.3
Right Side 8.2 17.9 7.8 22.7 8.5 6.6
-+ 1.4 _+ 2.2 -+ 1.6 __ 7.6 -+ 1.3 -+ 0.7
Left Side 6.9 23.0 8.3 18.1 8.9 6.8
-+ 0.7 -+ 4.4 -- 1.6 -+ 7.4 -+ 1.1 -+ 1.0
Serotonin Right Side 24.8 26.3 15.8 26.0 5.4 7.4
_+ 3.0 _+ 3.2 _+ 1.8 _+ 2.0 -+ 0.7 -+ 0.6
Left Side 22.6 26.7 16.4 23.9 5.5 7.8
-+ 3.1 _+ 3.5 -+ 2.4 - 2.1 _+ 0.6 _+ 0.7
Values are the mean (-+S.E.M.) of monoamine concentration (ng/mg protein). *p=0.015 ipsi- vs. cant differences between right and left catecholamines concentration in the brain areas studied. The paired t-test shows: DA striatum, t =0.38, p =0.7148; DA s. nigra, t = -0.36, p =0.7237; DA n. accumbens, t = - l . 1 4 , p=0.2777; DA VTA, t=0.22, p=0.8327; DA frontal lobe, t = - 0 . 0 7 , p=0.9492; DA hippocampus, t=0.98, p=0.3490; NA striatum, t = - 0 . 2 1 , p=0.8421; NA s. nigra, t = 1.29, p=0.2335; N A n . accumbens, t = l . 2 0 , p=0.2570; NA VTA, t = - 1 . 4 2 , p=0.1930; NA frontal lobe, t = - 0 . 4 5 , p =0.6609; NA hippocampus, t = - 0 . 1 5 , p =0.8856; 5-HT striatum, t = - 0 . 2 1 , p =0.8399; 5-HT s. nigra, t=0.62, p=0.5524; 5-HT n. accumbens, t=0.89, p=0.3936; 5-HT VTA, t = - 0 . 2 1 , p=0.8369; 5-HT frontal lobe, t = - 0 . 1 5 , p =0.8862; 5-HT hippocampus, t = - 0 . 6 4 , p =0.5372.
TABLE 2 DOPAMINE, NOREPINEPHRINE AND SEROTONIN LEVELS IN THE IPSI- AND CONTRA-LATERAL BRAIN SIDE RELATED TO THE PREFERRED DIRECTION IN THE T-MAZE TEST Dopamine IpsiLateral S. Nigra VTA Striatum Accumbens Frontal L Hippocampus
9.2 15.4 36.0 69.0 2.2 1.5
_+ 0.9 _+ 1.9 _+ 12.2 _+ 8.8 -+ 0.6 _+ 0.4
Norepinephrine
ContraLateral 9.9 15.6 32.3 73.8 2.2 0.6
-+ 0.9 -4- 2.6 -+ 10.6 -+ 10.5 _+ 0.9 -+ 0.2
IpsiLateral 8.0 17.7 7.2 22.7 8.5 6.6
-+ 0.4 -+ 2.2 _+ 1.6 _+ 7.6 -+ 1.3 _+ 0.4
ContraLateral 7.0 23.1 8.8 18.1 8.8 6.7
-+ 0.7 --- 4.3 -+ 1.6 -+ 4.1 -+ 1.1 -+ 0.5
Serotonin IpsiLateral 24.7 26.3 15.8 25.9 5.4 7.1
_+ 3.0 _+ 3.2 -+ 1.8 -+ 2.0 _+ 0.7 -+ 0.5
ContraLateral 22.6 26.7 16.4 23.9 5.4 8.0
-+ 3.1 _+ 3.5 _+ 2.4 -+ 2.1 _+ 0.6 -+ 0.7
Values are the mean (-+S.E.M.) of monoamine concentration (ng/mg protein). *p=0.016 ipsi- vs. contra-lateral side, t(l 1)=2.85, for the hippocampus. There were no significant differences for the other brain areas studied. The paired t-test shows: DA striatum, t=0.56, p=0.5892; DA s. nigra, t = - 0 . 6 3 , p=0.5435; D A n . accumbens, t = - 0 . 8 5 , p=0.4145; DA VTA, t = - 0 . 0 9 , p=0.9292; DA frontal lobe, t=-0.04,p =0.9672; NA striatum, t = - 0 . 7 5 , p = 0 . 4 7 4 2 ; NA s. nigra, t =0.99,p=0.3516; N A n . accumbens, t = 1.19, p =0.2601; NA VTA, t = - 1.55, p=0.1594; NA frontal lobe, t = - 0 . 2 9 , p =0.7765; NA hippocampus, t=-O. 19, p=0.8527; 5-HT striatum, t = - 0 . 2 2 , p=0.8397; 5-HT s. nigra, t=0.63, p=0.552; 5-HT n. accumbens, t=0.88, p=0.396; 5-HT VTA, t = - 0 . 2 3 , p=0.8362; 5-HT frontal lobe, t = -0.13, p =0.88; 5-HT hippocampus, t = - 1.41, p =0.1862.
s h o w f i g h t - p r e f e r e n c e . T h e s e r e s u l t s are c o n s i s t e n t w i t h t h e p o p u l a t i o n f i g h t - b i a s e d l a t e r a l i z a t i o n in t h e T - m a z e t h a t w e h a v e r e c e n t l y r e p o r t e d [6]. T h e r e s u l t s o f b i o c h e m i c a l a n a l y s i s in the s t r i a t u m , acc u m b e n s , f r o n t a l lobe, s. nigra, v e n t r a l t e g m e n t a l a r e a a n d h i p p o c a m p u s a p p e a r s in T a b l e 1 (for fight a n d left b r a i n side) a n d T a b l e 2 (for t h e T - m a z e p r e f e r r e d direction). T h e s e d a t a d o n o t d e m o n s t r a t e a significant d i f f e r e n c e in n o r e p i n e p r h i n e o r s e r o t o n i n l e v e l s b e t w e e n v a l u e s o f b r a i n sites c o n t r a l a t e r a / t o t h e T - m a z e p r e f e r r e d d i r e c t i o n c o m p a r e d to v a l u e s for t h e ipsilateral b r a i n sites. D o p a m i n e v a l u e s w e r e t h e s a m e for t h e b r a i n a r e a s s t u d i e d e x c e p t f o r t h e h i p p o c a m p u s , f o r w h i c h a m a r k e d a s y m m e t r y w a s f o u n d (Table 2). T h e
d o p a m i n e c o n c e n t r a t i o n w a s significantly h i g h e r for t h e b r a i n side ipsilateral to t h e T - m a z e c h o i c e , t ( 1 1 ) = 2 . 8 5 , p = 0 . 0 1 5 9 . T h e r e are n o statistical d i f f e r e n c e s b e t w e e n t h e right a n d left side in the b r a i n a r e a s s t u d i e d (Table 1). DISCUSSION T h e d e m o n s t r a t i o n o f f u n c t i o n a l a s y m m e t r i e s in a n i m a l s a p p e a r s to s u p p o r t t h e h y p o t h e s i s t h a t biologically determined asymmetries have persisted throughout vertebrate e v o l u t i o n , a n d t h a t h u m a n a s y m m e t r i e s are o n l y a n e x a m p l e of a fundamental feature of vertebrate neuroanatomy and b e h a v i o r [13]. P r e v i o u s l y , we h a v e r e p o r t e d a m a r k e d
788
DIAZ PALAREA, G O N Z A L E Z AND RODRIGUEZ
right-biased lateralization in the T-maze test at both individual and population levels (85.7% of the rats presented a behavioral asymmetry, 80% being right-biased within the lateralized group). In the present study, we report that spatial asymmetry in the T-maze is related to an imbalance between left and right DA levels of the hippocampus. Initially, a histochemical study suggested that the presence of dopamine in the hippocampus is independent of noradrenergic neurons [20]. However, other authors assumed that hippocampal DA only serves as the precursor to norepinephrine [5,22]. Evidence for a transmitter role of dopamine in the rat hippocampal formation has been provided more recently [3, 21, 26]. Thus, several studies reported the existence in the hippocampus of both a DA-sensitive receptor site [2] and DA-sensitive adenylate cyclase [12]. Moreover, haloperidol and apomorphine induce biochemical modification in the hippocampus similar to that observed in other DA-rich brain areas. The 6hydroxydopamine-induced destruction of ascending noradrenergic pathways failed to affect hippocampal DOPAC levels and only slightly reduced DA content in comparison with the almost total depletion of norepinephrine [3]. In conclusion, the previous biochemical findings suggest the existence of DA-synapses in the hippocampal formation. This hypothesis is also supported by the previous study of
Ishikawa et al. [21] who reported differences in the intrahippocampal distribution of norepinephrine and dopamine. The present experiment revealed that the interhemispheric balance of catecholamines in the hippocampus is different for dopamine and norepinephr~me, and therefore is a new evidence that hippocampal DA is independent of the noradrenergic system. The hippocampus is a brain structure previously related to various functions including spatial organization of behavior [1,4]. In spite of the reported morphological [11,10], biochemical [30] and functional [29] asymmetries in the hippocampus of nonhuman species, the possible role of DA-innervation has not been previously evaluated. In the present study we report a new biochemical asymmetry for the hippocampus related to dopamine innervation. In addition, our data strongly suggest that this asymmetric distribution of dopamine is related to the spatial organization of behavior in the T-maze test. As shown in Table 2, the DA levels in the homolateral hippocampus to the T-maze spatial preference is greater than in the contralateral side. In the present study, the NA/DA and 5-HT/DA ratios are similar to those previously reported by Ishikawa et al. [24]. The NA or 5-HT determinations reveal an interhemispheric balance in the brain areas studied, including the hippocampus. Therefore, we conclude that in the hippocampus, DA levels and not NA or 5-HT levels are related to spatial preference.
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