EFFECTS OF AMYGDALOID LESIONS ON PLASMA AND PITUITARY LEVELS OF LUTEINIZING HORMONE IN THE MALE DEERMOUSE

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1 EFFECTS OF AMYGDALOID LESIONS ON PLASMA AND PITUITARY LEVELS OF LUTEINIZING HORMONE IN THE MALE DEERMOUSE B. E. ELEFTHERIOU, A. J. ZOLOVICK and R. L. NORMAN Department of Zoology, Kansas State University, Manhattan, Kansas, U.S.A. (Received 23 January 1967) SUMMARY Bilateral lesions produced by electrocoagulation in the basolateral amygdaloid nuclear complex were found to affect plasma and pituitary levels of luteinizing hormone (LH) in the male deermouse (Peromyscus maniculatus bairdii). Within 1 week after lesions were produced in the basolateral amygdaloid group, pituitary levels of LH increased significantly by 170% from the level in intact controls of 0\m=.\17\p=n-\0\m=.\47m-u./mg. During the same period, plasma levels rose by approximately 220% from 0\m=.\14\p=n-\0\m=.\45 m-u./ml. The rise in LH continued and was at 0\m=.\55m-u./mg. in the pituitary and 0\m=.\54m-u./ml. in plasma 3 weeks after the lesions had been placed. The rise in plasma LH was reflected in the weight of the testes, prostates and seminal vesicles which significantly increased in all animals with lesions. INTRODUCTION The regulatory role of the hypothalamus in the secretion of hypophysial hormones is well established. Several investigations, however, indicate that other areas of the brain are also involved. More specifically, the amygdala, a part of the rhinencephaliclimbic system, has been implicated in modulating gonadotrophic and corticotrophic activity (Koikegami, Yamada & Usui, 1953 ; Koikegami, Fuse, Yokoyama, Watanabe & Watanabe, 1955 ; Shealy & Peele, 1957 ; Alonso-De Florida & Delgado, 1958 ; Mason, 1959; Eleftheriou, Zolovick & Pearse, 1966). More recently, it was found that lesions placed in the basolateral amygdaloid complex result in the continuous release of luteinizing hormone (LH) from the pituitary of the female deermouse (Eleftheriou & Zolovick, 1967). The present report deals with the effects of lesions placed in the basolateral amygdaloid complex on pituitary and plasma levels of luteinizing hormone and on the weight of testes, seminal vesicles and prostates in the male deermouse (Peromyscus maniculatus bairdii). * Contribution no. 388, Department of Zoology, Kansas Agricultural Experimental Station, Manhattan, Kansas, U.S.A.

2 Materials and methods Adult male deermice, weighing g., were anaesthetized with sodium pentobarbitone and oriented in a stereotaxic instrument ; lesions were then placed bilaterally by thermocoagulation in the basolateral nuclear group of the amygdaloid complex according to stereotaxic atlas for this species (Eleftheriou & Zolovick, 1965). A large stainless-steel bar, inserted in the rectum, served as the indifferent electrode. Three groups of ten animals each were killed 3 days and 1, 2 and 3 weeks (30 animals/period) after the lesions had been produced. Three similar groups of nonlesioned controls were also killed. An additional three groups were sham-operated by inserting the needle into the amygdala without administering current. The last three groups were killed 1 week after the operation. The entire experiment was conducted during the month of September. Fig. 1. Diagrammatic representation of the diencephalon of P. m. bairdii. Meshed area on left hemisphere represents location of lesions. Right hemisphere represents location of various nuclei : (1) ABL = basolateral nucleus; (2) AME = medial amygdaloid nucleus; (3) ACE = central amygdaloid nucleus; (4) AL = lateral amygdaloid nucleus; (5) AGO cortical amygdaloid = nucleus. At the time of killing, blood was collected in heparinized syringes by entering the orbital sinus of the eye. Plasma samples were obtained by centrifugation at 1000g for 15 min. and frozen for later analyses. Pituitaries were removed, weighed to the nearest 0-1 mg. and frozen in saline. In addition, testes, seminal vesicles and prostates were also removed, cleaned of extraneous tissue and weighed to the nearest 0-1 mg. on a torsion balance. The position of lesions was confirmed by histological examina tion (Fig. 1). Pituitary and plasma levels of luteinizing hormone were estimated by the ovarian ascorbic acid depletion method (Parlow, 1961). Immature, Holtzman strain, female rats, weighing g., served as receptor animals for the pituitary homogenates and plasma samples. One ml. of pituitary homogenate (3 mg./ml.) from each of a group of ten male deermice was injected into groups of three rats and 0-8 ml. plasma was injected into two rats. In addition, 0-9% NaCl solution and 1,5, 10 and 20 fig.

3 - - NIH-S-5 ovine standard LH was injected into 16 rats, each dose into four rats, to establish a standard regression curve for LH. Pituitary and plasma LH were calcu lated from a standard log curve derived from the following equation: y log x, where y is OAA and x is fig. LH. The values for LH were then trans formed into m-u. LH/mg. pituitary tissue or m-u. LH/ml. plasma. Analysis ofvariance and i-tests were used to test significance. Light and diet were not altered throughout the experiments. The body weight of the animals remained much the same. RESULTS The limits of the lesions in the anterior-posterior direction extended from the first one-third of the dorsal and ventral hypothalamic nuclei posteriorly to the level of Pituitary 0-4 h c _L 1 2 Time after lesions (weeks) Fig. 2. Plasma and pituitary levels of luteinizing hormone (in m-u./mg. or ml.) in intact, control, male P. m. bairdii and in animals with lesions in the basolateral amygdaloid complex after 3 days and 1, 2 and 3 weeks. Plasma (Sp ) and pituitary (Sp) levels of LH in sham-operated animals are also indicated.

4 the anterior limits of the posterior hypothalamic nuclei. Laterally, the lesions extended from the middle of the basolateral amygdaloid nuclei to the middle of the lateral of about amygdaloid nuclei. The lesions were cylindrical with an average height 0-8 mm. and a diameter of 0-7 mm. No lesion exceeded these boundaries. Within 1 week after lesions had been placed in the basolateral amygdaloid complex, the pituitary level of LH rose significantly (P < 0-01) from 0-17 m-u./mg. in intact, control deermice to 0-47 m-u./mg. (Fig. 2). An accompanying significant (P < 0-01) rise in the plasma level of LH from 0-14 to 0-45 m-u./ml. was also found during this period. The pituitary and plasma levels of LH continued to rise significantly (P < 0-001) until 3 weeks after the lesion the levels were 0-55 m-u./mg. and 0-54 m-u./ ml., respectively. The plasma levels of LH in sham-operated animals showed a small, non-significant rise to 0-21 m-u./ml. The pituitary levels of LH in sham-operated animals rose to 0-33 m-u./mg., but this increase was not statistically significant due to great variations. Table 1. Weights of testes, seminal vesicles and prostates in sham-operated and intact male Peromyscus maniculatus bairdii and in animals with lesions in the basolateral amygdaloid complex at 3 days and 1, 2 and 3 weeks (means ± S.E.) mg./100 g. body weight Time,-*-, Treatment after lesion Testes Seminal vesicles Prostates Intact 1198± Lesions 3 days ± ± week 1335± ± ± weeks ± weeks 1363± ±28-4 Sham-operated 1 week 1222± ± The weight of the testes in animals with lesions increased by 11 and 14 % (Table 1) during the first and second weeks, respectively ; but this change was not a significant increase (P > 0-1). In contrast, the weight of the seminal vesicles increased by a significant (P < 0-05) 32% from in controls to mg./100 g. body weight 1 week after lesions had been produced (Table 1 ). Thereafter, the weight ofthese organs increased further to mg./100 g. at 2 weeks (P < 0-01) and then decreased to mg./100 g. at 3 weeks, a value not significantly (P > 0-10) different from those in intact controls. The weight of the prostate glands in deermice with lesions increased significantly (P < 0-05) after 1 week from 83-5 in intact controls to mg./100 g. (Table 1). The prostate glands at 2 weeks showed an increase of 60 % which was significant (P < 0-01). Three weeks after lesions had been produced, the prostate glands in creased by 49 % from a value of 83-5 in the controls to mg./100 g. Shamoperated animals showed no significant changes in organ weights (Table 1). Variability in organ weights and LH values occurred in all groups regardless of treatment, owing to the fact that our colony of Peromyscus maniculatus bairdii is not inbred and therefore non-homogeneous. The wide variability in the sham-operated controls can be partially explained if one assumes that the area of the amygdala which modulates LH synthesis and release

5 strikes this area it of the critical area. In effect these animals will be 'lesioned'. is small and well delineated. Thus, if the electrode inadvertently will result in damage DISCUSSION Although further results are necessary to complete the elucidation of the mechan ism by which lesions in the basolateral amygdaloid complex affect changes in pituitary and plasma levels of LH, the present findings, in the male deermouse, together with previous work in female animals (Yamada & Greer, 1960; Shealy & Peele, 1957; Koikegami et al. 1953; Koikegami et al. 1955; Eleftheriou & Zolovick, 1967), indicate the existence of a separate inhibitory centre, in both sexes, for the regulation of luteinizing hormone. Both the pituitary and plasma levels of LH in the male deermouse were significantly lower (by a factor of 3-6 and 5-8, respectively) compared with the female deermouse (Eleftheriou & Zolovick, 1967).Thusatdioestrus, the pituitary LH content in the female deermouse was 0-62 m-u./mg. as compared to 0-17 m-u./mg. in the male. The plasma levels during the same periods in the two sexes were 0-82 and 0-14 m-u./ml., respec tively. Within 3 weeks after lesions had been placed in the basolateral amygdaloid complex, the plasma level of LH in the female was 2-63 m-u./ml. but only 0-54 m-u./ ml. in the male. It seems, therefore, that the lesions produced in the basolateral amygdaloid complex caused a proportionately greater synthesis and release of LH in the pituitary of the female deermouse than in the pituitary of the male deermouse. There is also the possibility that the injury produced a focal point of irritation which was the starting point of potentials that spread to other areas of the brain, and mainly to the hypothalamus (Taleisnik, Caligaris & DeOlmos, 1962) for the release of LH. However, it seems unlikely that such an irritation would have produced sustained secretion of LH over a period of 3 weeks. Animals which were sham-operated by placing electrodes in the amygdala but without administration of current, did not show significant changes in LH or in the weight of accessory sex organs. This finding is in agreement with previous observa tions (Bunn & Everett, 1957 ; Eleftheriou & Zolovick, 1967) that mere insertion of an electrode does not act as a stimulus for the release of LH. This work was supported by grant no. HD from the Child Health and Human Development Council of the National Institutes of Health, and GM-32,159 from the National Institute of General Medical Sciences, Bethesda, Maryland. NIH-LH, ovine S-5 Lot no A was obtained from the Endocrinology Section of NIH, Bethesda, Maryland. REFERENCES Alonso-De Florida, F. & Delgado, J. M. R. (1958). Lasting behavioral and EEG changes in cats induced by prolonged stimulation of amygdala. Am. J. Physiol. 193, Bunn, J. P. & Everett, J. W. (1957). Ovulation in persistent estrous rats after electrical stimulation of the brain. Proc. Soc. exp. Biol. Med. 96, Eleftheriou, B. E. & Zolovick, A. J. (1965). The forebrain of the deermouse in stereotaxic coordinates. Kans. Agr. Exp. Sta. Tech. Bull. no Eleftheriou, B. E., Zolovick, A. J. & Pearse, R. (1966). Effect of amygdaloid lesions on the pituitaryadrenal axis in the deermouse. Proc. Soc. exp. Biol. Med. 122,

6 Eleftheriou, B. E. & Zolovick, A. J. (1967). Effect of amygdaloid lesions on plasma and pituitary levels of luteinizing hormone. J. Reprod. Fert. (In Press.) Koikegami, H., Yamada, T. & Usui, K. (1953). Stimulation of amygdaloid nuclei and periamygdaloid cortex with special reference to its effects on uterine movements and ovulation. Folia psychiat. neural. jap. 8, Koikegami, H., Fuse, S., Yokoyama, T., Watanabe, T. & Watanabe, H. (1955). Contribution to the comparative anatomy of the amygdaloid nuclei of mammals with some experiments of their destruction or stimulation. Folia psychiat. neurol. jap. 8, Mason, J. W. (1959). Plasma 17-hydroxycorticosteroid levels during electrical stimulation in amygdaloid complex in conscious monkeys. Am. J. Physiol. 196, Parlow, A. F. (1961). Bioassay of pituitary luteinizing hormone by depletion of ovarian ascorbic acid. In Human pituitary gonadotropins, p Ed. A. Albert. Springfield, Illinois: C. C. Thomas. Shealy, N. C. & Peele, T. L. (1957). Studies on amygdaloid nucleus of the cat. J. Neurophysiol. 20, Taleisnik, S., Caligaris, L. & DeOlmos, J. (1962). Luteinizing hormone release by cerebral cortex stimula tion in rats. Am. J. Physiol. 203, Yamada, T. & Gréer, M. A. (1960). The effect of bilateral ablation of the amygdala on endocrine function in the rat. Endocrinology 66,