Neuroscience Letters, 116 (1990) 320 324 Elsevier Scientific Publishers Ireland Ltd

320

NSL 07079

Biphasic effects of polarizing current on adenosine-sensitive generation of cyclic AMP in rat cerebral cortex Yukio Hattori, Akiyoshi Moriwaki and Yasuo Hori

Department of Physiology, Okayama University Medical School, Okayama (Japan) (Received 8 November 1989; Revised version received 9 March 1990; Accepted 27 April 1990)

Key words. Anodal polarization; Direct current; Cyclic AMP; Adenosine; Brain slice; Cerebral cortex; Rat Cyclic AMP accumulation elicited by adenosine was investigated in cortical slices of rats following the application of a weak anodal direct current (anodal polarization) to the unilateral sensorimotor cortex. Anodal polarization with 3.0 #A for 30 min caused an increase in the adenosine-elicited accumulation of cyclic AMP in the polarized cortex, in which case the increase in the polarized cortical region was highlighted by repeated applications of the currents, Polarization with 0.3/tA for 30 min decreased the cyclic AMP accumulation, and polarization with 30.0/~A for 30 min had no effect. When applied for 3 h, the polarizing currents at all of the intensities tested decreased the cyclic AMP accumulation. The results indicate that anodal polarization has biphasic effects on adenosine-elicited accumulation of cyclic AMP in the cortex. Alterations in the cyclic AMP generation are suggested to form the neurochernical basis of central and behavioral activity induced by anodal polarization.

A p p l i c a t i o n o f a w e a k a n o d a l direct c u r r e n t ( a n o d a l p o l a r i z a t i o n ) to restricted cortical areas, such as m o t o r cortex, has been s h o w n to cause characteristic c h a n g e s in e l e c t r o p h y s i o l o g i c a l activity o f the c o r t e x a n d p e r i p h e r a l m a n i f e s t a t i o n s [3, 10, 11]. S u b s e q u e n t findings have revealed t h a t the effects o f a n o d a l p o l a r i z a t i o n o n behavioral m o t o r m a n i f e s t a t i o n s last for several weeks o r m o r e in r a b b i t s [8]. The p h e n o m e n o n has been i m p l i e d to be due to f o r m a t i o n o f a c h r o n i c excitation focus, which is called d o m i n a n t focus, at the p o l a r i z e d point. It has been s h o w n t h a t a d e n o s i n e exerts its effect in s y n a p t i c t r a n s m i s s i o n in the central n e r v o u s system [15], a n d t h a t a d e n o s i n e has a p o t e n t effect o n eliciting cyclic A M P a c c u m u l a t i o n in b r a i n slice p r e p a r a t i o n s o f several species o f a n i m a l s [4]. The p h y s i o l o g i c a l a c t i o n s o f a d e n o s i n e have led to the s p e c u l a t i o n t h a t adenosine-sensi-

Correspondence." Y. Hattori, Department of Physiology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

321 tive cyclic AMP-generating systems may be altered in the polarized cortex. In view of previous findings [7], one episode of anodal polarization is presumed to leave some traces involving activity of cyclic AMP generation in the cortex, especially in the polarized cortical region, even before the stable motor manifestations, for which repeated applications of polarizing currents are necessary, are achieved. To clarify this possibility, in the present study, cyclic AMP accumulation elicited by adenosine was examined in slices of polarized and non-polarized cortical regions of rats after 1 to 5 polarization trials were delivered to the unilateral sensorimotor cortex. Male Wistar rats weighing 19(~240 g were anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/kg). Silver electrodes of 1 mm in diameter were bilaterally implanted in the cranial bone over the sensorimotor cortex so as to set the tip on the dura mater. Another indifferent electrode was situated on the midline of the nasal bone. One week or more after the surgery, anodal direct currents of 0.3, 3.0 or 30.0/~A were applied for 30 min or 3 h to the left sensorimotor cortex using the indifferent electrode under unrestrained, unanesthetized conditions. When required, anodal polarization (3.0/tA, 30 min) was repeated 3 or 5 times once a day for successive days. No current was applied for control rats. Rats were killed by decapitation on the day following termination of anodal polarization, and the cerebrum was quickly extirpated. The cortical region including the polarized point (left) and the contralateral cortical region (right) were dissected from the anterior cortex and cross-chopped to 400/~m in thickness with a McIlwain tissue chopper. In control rats, cortical slices of the corresponding regions were prepared in the same way at comparable time periods. After a 60-min preincubation, the slices (approximately 3 mg protein) of each cortical region were incubated for 10 rain in 5 ml of K r e b ~ R i n g e r bicarbonate-glucose solution with or without 0.1 mM adenosine (Sigma). The solution contained (in mM): NaC1 118, KC1 4.7, CaC12 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and glucose 10. All of the preincubations and incubations were carried out at 37'~C with constant aeration with 95% 02 and 5% CO2. At the end of the incubation, 2.5 ml of ice-cold 7% trichloroacetic acid was added to the slices, and the mixture was homogenized in an ice bath. After purification by Dowex 50W-X8 column chromatography, cyclic AMP was assayed using assay kits available from Amersham International. Protein content of the homogenate was determined by the method of Lowry et al. [9]. Cyclic AMP contents are expressed as picomoles per milligram of protein. Statistical significance was evaluated by analysis of variance followed by Student's t-test. Fig. 1 shows the cyclic AMP contents of incubated cortical slices of rats in which a polarizing current (3.0/IA, 30 min) was applied 5 times or less to the left sensorimotor cortex. The basal levels of cyclic AMP did not vary between the polarized and non-polarized control rats throughout the polarization trials. The cyclic AMP contents were increased 12- to 19-fold by the addition of 0.1 mM adenosine. Anodal polarization with 3.0 /~A increased the adenosine-elicited accumulation of cyclic AMP. After one trial of polarization, a significant increase in cyclic AMP accumulation was observed in both the polarized and non-polarized cortical regions, but the cyclic AMP levels were somewhat higher in the polarized region than in the non-

322

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o , ~ ~ , ? 0 1 2 3 4 5 0 1 2 3 4 5 Number of polarization

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Fig. I. Cyclic A M P contents in cortical slices incubated with or without adenosine after application of polarizing current or no current. Anodal direct current of 3.0/~A was applied for 30 min to the left sensorimotor cortex, and the application was repeated three or five times once a day. Slices of the left (A) and right (B) cortical regions were prepared from the anterior cortices of the polarized and non-polarized control rats. The slices were incubated with ( 0 ) or without ( O ) 0.1 m M adenosine. Each value represents the mean + S.E.M. o f 7-11 different rats. *Significantly different (P < 0.05) from the value of non-polarized control rats.

polarized region. During repetition of polarization trial, the cyclic AMP levels remained almost constant in the polarized region, whereas in the non-polarized region they returned to the levels before polarization. Fig. 2 shows the effects of one polarization trial with different intensities (0.3, 3.0, and 30.0/,A) or durations (30 min and 3 h) on the adenosine-elicited accumulation of cyclic AMP in the left cortical region. Under these conditions, the cyclic AMP contents were increased 6- to 17-fold by the addition of 0.1 mM adenosine. When applied for 30 min, the polarizing current of 3.0/aA invariably produced the significant increase in adenosine-elicited accumulation of cyclic AMP. Conversely, a cur-

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B F,

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0.3

3.0

30.0

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0.3

3.0

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Polarizing current (pA) Fig. 2. Effect of polarizing current with different intensities or durations on adenosine-elicited accumulation of cyclic A M P in cortical slices. Anodal direct current of 0.3, 3.0 or 30.0 p A was applied for 30 min (A) or 3 h (B) to the left sensorimotor cortex. Slices o f the left cortical region were prepared from the anterior cortices o f the polarized and non-polarized control rats, and incubated with 0.'1 m M adenosine. Each value represents the mean + S.E.M. o f 6-8 different rats. *Significantly different ( P < 0.05) from the value of non-polarized control rats.

323 rent of 0.3 pA significantly decreased the cyclic AMP accumulation. A current of 30.0 pA had no effect. When applied for 3 h, the polarizing currents at all of the intensities tested resulted in a significant decrease in the cyclic AMP accumulation. Separate experiments showed that adenosine elicited the cyclic AMP accumulation in a dose-dependent manner, and that the elicitation by adenosine of cyclic AMP accumulation was blocked by an adenosine antagonist, 8-phenyltheophylline. In agreement with the previous findings on cortical slices from rats [12-14], the present results indicate that adenosine markedly elicited cyclic AMP accumulation. In this study, it should be noted that the adenosine-elicited accumulation of cyclic AMP fluctuated in the polarized cortex. It has been shown that adenosine exerts several effects including the regulation of synaptic transmission in the central nervous system through cell surface adenosine receptors [15]. Further, adenosine has been implicated in mechanisms of behavioral changes in several species of animals [6]. Concerning the actions of adenosine, the cyclic AMP generation through adenosine receptor-coupled adenylate cyclase system has been extensively studied using brain slice or cell-free preparations [1, 2, 5]. In our experiments with polarized cortex, the effects of adenosine on cyclic AMP accumulation were dose-dependent and were blocked by the adenosine antagonist 8-phenyltheophylline. Thus, it is likely that polarizing currents alter the adenosine receptor-mediated cyclic AMP generation in the cortex. The fluctuation by anodal polarization of adenosine-elicited accumulation of cyclic AMP depended on both the intensity and duration of polarizing currents; in most cases, there was a tendency for the currents to decrease the cyclic AMP accumulation, but the polarization with 3.0 pA for 30 rain profoundly increased cyclic AMP accumulation. The biphasic effects of anodal polarization may indicate that the currents have two opposite actions on neuronal activity of the cortex, although the evaluation of this possibility requires direct electrophysiological analyses. It has been shown that current intensities of around 3.0 pA are optimum for the establishment of peripheral motor behavior, and that higher and lower current intensities are inconvenient or have rather disturbing effects [8]. Taken together, the present results seem to provide neurochemical evidence for the optimum intensity of polarizing currents in establishing the behavioral manifestations induced by anodal polarization. In a previous study on the cortex of rabbits which showed characteristic motor responses to extraneous stimuli resulting from repeated applications of polarizing currents, it was found that there was a regional difference in cyclic AMP accumulation [7]. The present results clearly show that a similar difference in the adenosineelicited accumulation of cyclic AMP between the polarized and non-polarized regions appeared during repeated applications of polarizing currents, although the bilateral effects of the currents observed only after one trial of polarization remain to be examined, Thus, it is conceivable that the repetition of anodal polarization makes regional differences in cyclic AMP generation sharp and leads to the establishment of behavioral manifestations. In conclusion, the application of polarizing currents to the restricted cortical point was found to alter the cyclic AMP accumulation elicited by adenosine. The effects

324 o f a n o d a l p o l a r i z a t i o n were biphasic: s t i m u l a t o r y a n d i n h i b i t o r y to cyclic A M P g e n e r a t i o n . T h e s t i m u l a t o r y effects were o b s e r v e d w h e n the p o l a r i z i n g c u r r e n t s o f 3 . 0 / z A were a p p l i e d , a n d r e p e a t e d a p p l i c a t i o n s o f the c u r r e n t s were significant in h i g h l i g h t i n g their effects o n the cyclic A M P a c c u m u l a t i o n in p o l a r i z e d cortex. T h u s , it is likely t h a t the a l t e r a t i o n s in a d e n o s i n e - s e n s i t i v e g e n e r a t i o n o f cyclic A M P p l a y a f u n d a m e n t a l role in the n e u r o c h e m i c a l m e c h a n i s m s o f c o r t i c a l e x c i t a t i o n i n d u c e d b y a n o dal p o l a r i z a t i o n . 1 Anand-Srivastava, M.B. and Johnson, R.A., Regulation of adenosine-sensitiveadenylate cyclase from

rat brain striatum, J. Neurochem., 35 (1980) 905-914. 2 Bazil, C.W. and Minneman, K.P., An investigation of the low intrinsic activity of adenosine and its analogs at low atfinity (A2) adenosine receptors in rat cerebral cortex, J. Neurochem., 47 (1986) 547~553. 3 Bindman, L.J., Lippold, O.C.J. and Redfearn, J.W.T., Long-lasting changes in the level of the electrical activity of the cerebral cortex produced by polarizing currents, Nature, 196 (1962) 584-585. 4 Daly, J.W., Cyclic Nucleotides in the Nervous System, Plenum, New York, 1977, 401 pp. 5 Daly, J.W., Butts-Lamb, P. and Padgett, W., Subclasses of adenosine receptors in the central nervous system: interaction with caffeine and related methylxanthines, Cell. Mol. Neurobiol., 3 (1983) 69-80. 6 Dunwiddie, T.V., The physiological role of adenosine in the central nervous system, Int. Rev. Neurobiol., 27 (1985) 63-139. 7 Hattori, Y., Moriwaki, A., Pavlygina, R.A. and Hori, Y., Regional difference in the histamine-elicited accumulation of cyclic AMP in rabbit cerebral cortex with a cortical dominant focus, Brain Res., 279 (1983) 308-310. 8 Hori, Y. and Yamaguchi, K., Prolonged formation of a cortical dominant focus by anodal polarization, Med. J. Osaka Univ., 26 (1975) 27--38. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 10 Morrell, F., Effect of anodal polarization on the firing pattern of single cortical cells, Ann. N.Y. Acad. Sci., 92 (1961) 860--876, 11 Novikova, L.A., Rusinov, V.S. and Semiokhina, A.F., Electrophysiological analysis of closing function in the cerebral cortex of rabbit in the presence of a dominant focus, Zh. Vyssh. Nerv. Deiat., 2 (1952) 844-861. 12 Perkins, J.P. and Moore, M.M., Regulation of the adenosine cyclic 3', 5'-monophosphate content of rat cerebral cortex: ontogenetic development of the responsiveness to catecholamines and adenosine, Mol. Pharmacol., 9 (1973) 774-782. 13 RalI, T.W. and Sattin, A., Factors influencing the accumulation of cyclic AMP in brain tissue, Adv. Biochem. Psychopharmacol., 3 (t970) 113-133. 14 Schultz, J. and Daly, J.W., Accumulation of cyclic adenosine 3',5'-monophosphate in cerebral cortical slices from rat and mouse: stimulatory effect of ~- and fl-adrenergic agents and adenosine, J. Neurochem., 21 (1973) 1319-1326. 15 Snyder, S.H., Adenosine as a neuromodulator, Annu. Rev. Neurosci., 8 (1985) 103-124.

Biphasic effects of polarizing current on adenosine-sensitive generation of cyclic AMP in rat cerebral cortex.

Cyclic AMP accumulation elicited by adenosine was investigated in cortical slices of rats following the application of a weak anodal direct current (a...
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