Brain Research, 118 (1976) 503-508 @ Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

503

Effect ,of midbrain raphe nuclei lesions on the circadian rhythm of plasma corticosterone in the rat*

FRANK G. BALESTRERY and GARY P. MOBERG** Department of Animal Science, University of California, Davis, Calif. 95616 (U.S.A.)

(Accepted September 23rd, 1976)

Throughout the 24-h day the concentration of corticosteroids in plasma shows a definite circadian rhythmL The neural systems which are essential in transforming the environmental cues that entrain this rhythm have not been identified, however, evidence from limited studies has suggested that serotoninergic (5-hydroxytryptamine, 5-HT) systems projecting to the forebrain may be involved in modulating this rhythmic release of corticosterone. Correlations have been made between the rhythmic changes in whole brain or limbic structure 5-HT content in rats and the daily fluctuations of plasma corticosterone 14,17. Investigators have found that pharmacological disruption of the synthesis of 5-HT will alter the normal circadian rhythm ofplasmacorticosteroids. For example, Krieger and Rizzo 10 found in cats that various drugs which alter 5-HT melabolism were effective in blocking the normal daily rise in cortisol. Also, in rats it is found that blocking 5-HT synthesis with para-chlorophenylalanine (pCPA) not only results in decreased brain 5-HT content but also the abolishment of the circadian rhythm of plasma corticosteronel4.19. These data support the conclusion that 5-HT neurons are involved in the control of the circadian rhythm of the adrenal axis. In contrast to the above findings, Van Delft et al. is found that pCPA treatment in rats had only a partial effect on the circadian rhythm of corticosteroids and these authors suggested that 5-HT is not directly involved in the control of the adrenal rhythm but that the altered corticosterone levels reflect secondary effects of the drug. Certainly, pCPA not only inhibits the synthesis of 5-HT but also produces small depletions in brain catecholamines 7 as well as competing in the transport mechanisms for tryptophan, phenylalanine, and other amino acidsL Also, p C P A drastically alters many behavioral characteristics, causing insomnia, anorexia, and changes in patterns of motor activity, any of which may interfere with basal adrenal function, irrespective of an effect on the serotoninergic system per se. Therefore, to assess the role of the 5-HT neural system in the tonic control of the pituitary-adrenal axis without resorting to the use of drugs which may have non* Supported by NIH Grant NS09800. ** To whom reprint requests should be addressed.

504 specific and unwanted secondary effects, the plasma levels of corticosterone were measured in rats following ablation of the medial and dorsal raphe nuclei. Male Sprague-Dawley rats (from the U.C.D. colony) weighing 160-200 g at the time of surgery were maintained two per cage in the colony approximately 3 weeks prior to surgery. Throughout the experiment the animals received water and food ad libitum and were maintained at controlled temperature (22 ± 2 °C) on a light :dark 14:10 lighting schedule (lights on at 05.00 h). The animals were divided into 3 groups: unoperated controls, sham lesion controls, and animals with both the medial and dorsal raphe nuclei ablated. The animals to be lesioned were anesthetized with chloral hydrate (400 mg/kg) and a platinum tipped electrode was stereotaxically guided into the medial (A:0.4; D:2.3; L:0.0) and dorsal (A:0.4; D:4.3; L:0.0) raphe nuclei according to coordinates derived from the K6nig and Klippel s rat brain atlas. Each experimental animal was lesioned by passing 2 mA of direct anodal current for 10 sec through the electrode. The sham-lesioned animals received the same surgical treatment except that the electrode was lowered only 1.0 mm into the brain at the same location and no current was passed through the electrode. Six days post surgery at 05.00, 12.00, 19.00 and 24.00 h, groups of 12 animals were taken to an adjoining room and decapitated within 30 sec. Two such groups of 12 were sacrificed for each time period with a 2-3-day interval between each group. No personnel were allowed to disturb the animals 24-36 h prior to sacrifice. Trunk blood was collected into chilled heparinized tubes, centrifuged, and aliquots of plasma were frozen for subsequent analysis for corticosterone content by the fluorometric method of Givner and Rochefort 4. Following decapitation the brains were removed and the forebrain was separated by a dorsal-ventral section at the level of the posterior border of the hypothalamus, frozen on dry ice, and stored at --25 °C until the 5-HT content was determined by the method of Maickel et al. 11. The differences between means were tested for significance by the Student's t-test. The midbrain was fixed in 10 ~ formalin for histological evaluation. Fig. 1 shows an example of the lesions which were characterized as destroying the medial and dorsal raphe nuclei. In all animals the medial raphe was damaged over its entire rostrocaudal and dorsoventral extent. Additional damage from the medial lesions extended laterally into the reticular formation. Non-specific damage was usually found at the decussation of the brachium conjunctivum, rostrally, and the ventral tegmental nucleus caudally. No damage to the medial lemniscus was noted. The dorsal raphe lesions damage was confined mainly to the medial and caudal extent of the nucleus with rostral portions being the least damaged. Non-specific damage from the dorsal lesion was always found in the ventral and medial portions of the central gray and occasionally in the dorsal portions as well. The medial longitudinal fasciculus and ventral tegmental nucleus were usually damaged bilaterally. At rostral levels, the lII and 1V cranial nuclei were damaged. At medial and caudal levels, damage to the brachium conjunctivum was noted. As illustrated in Fig. 2, these lesions resulted in a highly significant reduction in the content of forebrain 5-HT at all times studied. However, as effective as the lesions were in reducing 5-HT levels, no alteration in the circadian rhythm of plasma cortico-

505

Fig. 1. Frontal section through the caudal mesencephalon illustrating the combined dorsal and medial raphe lesion. Cresyl violet stain. × 11. sterone was noted (Fig. 3). Nevertheless, ablation of these two major midbrain raphe nuclei did not prevent a small, but significant increase in forebrain 5-HT at 19.00 h. Such an increase might suggest that some other serotoninergic system may control the circadian rhythm of the adrenal axis. For example, the suprachiasmatic nuclei receive 5-HT projections and these nuclei are implicated in the control of circadian rhythms. However, Scapagnini et al. 14 found thatpCPA treatment, which abolished the circadian rhythm of plasma corticosterone and lowered the brain 5-HT to levels comparable to the present study, did not abolish circadian rhythm of brain 5-HT. Since it would be anticipated that the pCPA treatment would affect all serotoninergic systems equally, the remaining rhythm of 5-HT content observed in these two studies is not readily explained. Preliminary studies6,1~ had found that the resting level of plasma corticosterone, as measured at two times of day following raphe nuclei ablation, differed from normal values, suggesting that the raphe nuclei might be involved in the control of the normal corticosterone rhythm. In the present study when the levels of plasma corticosterone

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Fig. 2. The effect of combined medial and dorsal raphe lesions on levels of forebrain 5-HT throughout a 24-h period. Each bar represents the mean ± S.E.M. Numbers in parentheses are the number of animals per group. Since at no time there was a significant difference between sham and control animals, the means were combined. The 05.00 combined control mean was not significantly different from that of 24.00 (P > 0.05). All other intergroup comparisons of combined control means were highly significant (P < 0.001). Comparison of the means of the lesioned animals revealed no significant difference in 19.00 vs. 24.00 (P > 0.05), highly significant differences (P < 0.001) in 05.00 vs. 19.00, 12.00 vs. 19.00, and 12.00 vs. 24.00, and significant differences (P < 0.05) in 05.00 vs. 12.00 and 24.00. For all time periods, the lesion resulted in a highly significant (P < 0.001) reduction in forebrain 5-HT as compared to the combined control mean.

were measured t h r o u g h o u t the 24-h period, the a b l a t i o n of the raphe nuclei had n o effect o n the circadian r h y t h m even t h o u g h b r a i n 5-HT was reduced to a level comparable to the previous lesion studies. The reason for this discrepancy in findings is n o t readily apparent, however, spurious corticosteroid levels which do n o t reflect basal adrenal f u n c t i o n c a n be o b t a i n e d in animals that have n o t been well adapted to existing c o n d i t i o n s 16,20 whose corticosteroid levels are elevated up to 24 h prior to sacrifice ~ , or n o t decapitated rapidly, especially during the a.m. trough 1. I n the present study great care was t a k e n to o b t a i n samples that reflected the basal level of plasma corticosterone. Such precautions m a y be even more i m p o r t a n t in view of the finding that altering the levels of b r a i n 5-HT influences the adrenal response to stress 19. The earlier pharmacological studies using p C P A had f o u n d that the drug treatmerit appeared to alter the adrenal rhythm. However, Vernikos-Danellis et al. 19

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Fig. 3. Values of plasma corticosterone (mean 4_ S.E.M.) for the same animals represented in Fig. 2. The lesion, sham and control animals in each time period show no significant difference (P > 0.05) Therefore, all animals sacrificed at the same hour were combined and the combined means were compared. No significant difference (P > 0.05) exists between the combined means of 05.00 vs. 12.00. All other comparisons of combined means were highly significant (P < 0.001). found that the effect o f p C P A treatment was seen only 24 h after administration. Likewise Van Delft et al. is found that pCPA treatment only partially altered the circadian rhythm of plasma corticosterone and their data indicated that this alteration appeared to be the result of the drug influencing the adrenal sensitivity to ACTH. Our data would be in agreement with this conclusion since comparable reductions in brain 5-HT by raphe lesion do not alter the daily rhythmic changes of plasma corticosteroid levels. Other studies lend indirect support to our present findings. Dixit and Buckley 3 could not find a correlation between the rhythms of brain 5-HT and plasma corticosterone in adult rats. Okada 13 found that the maturation of the circadian rhythm of brain 5-HT is not comparable to the development of the pituitary-adrenal system's rhythm. Likewise, Krieger 9 found that permanent pharmacological disruption of the 5-HT system in neonatal rats had no effect on the circadian rhythm of plasma corticosterone in the adult. When rats are fed on a meal schedule that will alter the circadian rhythm of plasma corticosterone 12, the daily rhythm of 5-HT content in the limbic system is not affected (Moberg, unpublished). Thus the results of these studies and the present

508 findings on the effect of raphe lesions indicate that the serotoninergic system projecting from the midbrain is not essential in the control of the circadian rhythm of the adrenal axis and that any correlation between the circadian rhythms of these 5-HT systems and the adrenal axis is casual as opposed to causal.

1 Ader, R. and Friedman, S. B., Plasma corticosterone response to environmental stimulation. Effect of the duration of stimulation and the 24-hour adrenocortical rhythm, AnOn. Behav., 15 (1968) 37-44. 2 Critchlow, V., The role of light in the neuroendocrine system. In A. V. Nalbandov (Ed.), Advances in Neuroendocrinology, Univ. of Illinois Press, Urbana, lll., 1963. 3 Dixit, B. N. and Buckley, J. P., Circadian changes in brain 5-hydroxytryptamine and plasma corticosterone in the rat, Life Sci., 6 (1967) 755-758. 4 Givner, M. and Rochefort, J., An improved assay of corticosterone in rat serum and adrenal tissue, Steroids, 6 (1965) 486-489. 5 Grahame-Smith, D. G., Studies in vivo on the relationship between brain tryptophan, brain serotonin synthesis, and hyperactivity in rats treated with MAOI and L-tryptophan, J. Neurockem., 18 0971) 1053-1066. 6 Harrington, R. J., Scapagnini, U. and Moberg, G. P., Diurnal plasma B after raphe and septal lesions, J. Anita. Sci., 37 (1973) 313. 7 Koe, B. K. and Weissman, A., p-Chlorophenylalanine: a specific depletor of brain serotonin, J. PharmacoL exp. Ther., 154 (1966) 499-516. 8 K6nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain andLower Parts of the Brainstem, Williams and Wilkins, Baltimore, Md., 1963. 9 Krieger, D. T., Effect of intraventricular neonatal 6-OH dopamine or 5,6-dihydroxytryptamine administration on the circadian periodicity of plasma corticosteroid levels in the rat, Neuroendocrinology, 17 (1975) 62-74. 10 Krieger, D. T. and Rizzo, F., Serotonin mediation of circadian periodicity of plasma 17-hydroxycorticosteroids, Amer. J. Physiol., 217 0969) 1703 1707. I l Maickel, R. P., Cox, R. H., Saillant, J. and Miller, F. P., A method for the determination of serotonin and norepinephrine in discrete areas of the rat brain, Int. J. Neuropkarmacol., 7 (1968) 275-281. 12 Moberg, G. P., Bellinger, L. L. and Mendel, V. E., Effect of meal feeding on daily rhythms of plasma corticosterone and growth hormone in the rat, Neuroendocrinology, 19 (1975) 160-169. 13 Okada, F., The maturation of the circadian rhythm of brain serotonin in the rat, Life Sci., 10 (1971) 77 86. 14 Scapagnini, U., Moberg, G. P., Van Loon, G. R., De Groot, J. and Ganong, W. F., Relation of brain 5-hydroxytryptamine content to the diurnal variation in plasma corticosterone in the rat, Neuroendocrinology, 7 (1971) 90-96. 15 Scapagnini, U. and Preziosi, P., Role of brain NE and 5-HT in the tonic and phasic regulation of hypothalamic hypophyseal adrenal axis, Arch. int. Pharmacodyn., 196, Suppl. (1972) 205-220. 16 Seggie, J., Shaw, B., Uhlir, L. and Brown, G. M., Baseline 24-hour corticosterone rhythm in normal, sham-operated and septally lesioned rats, Neuroendocrinology, 15 0974) 51-61. 17 Simon, M. L. and George, R., Diurnal variations in plasma corticosterone and growth hormone as correlated with regional variations in norepinephrine, dopamine, and serotonin content of rat brain, Neuroendocrinology, 17 (1975) 125-138. 18 Van Delft, A. M. L., Kaplanski, J. and Smelik, P. G., Circadian periodicity of pituitary-adrenal function after p-chlorophenylalanine administration in the rat, J. Endocr., 59 (1973) 465-474. 19 Vernikos-Danellis, J., Berger, P. and Barchas, J. D., Brain serotonin and pituitary-adrenal function, Progr. Brain Res., 39 (1973) 301-308. 20 Wilson, M. and Critchlow, V., Effect of septal ablation on rhythmic pituitary-adrenal function in the rat, Neuroemlocrinology, 14 (1974) 333-344. 21 Zimmermann, E. and Critchlow, V., Suppression of pituitary-adrenal function with physiological levels of corticosterone, Neuroendocrinology, 5 (1969) 183-192.

Effect of midbrain raphe nuclei lesions on the circadian rhythm of plasma corticosterone in the rat.

Brain Research, 118 (1976) 503-508 @ Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands 503 Effect ,of midbrain raphe n...
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