Neonatal Stimulation and Maturation of the 24-Hour Adrenocortical Rhythm ROBERT ADER Department of Psychiatry, University of Rochester School of Medicine and Dentistry, Rochester, N.Y. 14642 (U.S.A.)
Experimentally imposed stimulation of the infant rat during the first 3 weeks of life is capable of modifying a variety of developmental processes, subsequent behaviors and physiological function, and ultimate susceptibility to experimentally induced organic disease (Ader, 1970; Denenberg, 1972). Based on the consistent finding that infant rats subjected to handling or electric shock stimulation show a reduced adrenocortical response to “stress” experienced later in life, several attempts have been made to relate adult adrenocortical and emotional reactivity to the release of steroids by the neonate in response to the stimulation or “stress” experienced during development (Levine and Mullins, 1966; Denenberg and Zarrow, 1971). A critical review of this literature has been presented in a previous volume in this series (Ader and Grota, 1973). Thus far the effects of early life experiences on subsequent adrenal function have been primarily concerned with adrenocortical reactivity. “Reactivity”, however, is a difficult dimension of adrenal function to define. Several studies (e.g., Zarrow et al., 1966, 1967; Ader et al., 1967; Friedman and Ader, 1967; Ader and Friedman, 1968) have shown that the adrenocortical response to environmental stimulation is determined by a complex interaction among several factors. These include the nature of the stimulus used to elicit an adrenal response, the duration or intensity of that stimulation, the time following stimulation when steroid samples are obtained, and the point in the 24-hr adrenocortical rhythm upon which the stimulation is superimposed. One might also question the criteria defining “reactivity”. Is “reactivity” reflected by the magnitude of the adrenocortical response, the latency of the responses and/or the duration of the elevation in steroid level? All of the above factors are relevant in defining and evaluating adrenocortical reactivity, particularly if one is interested in comparing differentially treated groups of animals. In view of these practical issues, studies of the neuroendocrine mediation of the behavioral and physiological effects of early life experiences become difficult to execute and to interpret. Many of these difficulties may be circumvented by studying the development of normal endocrine function. That is, by analyzing the effects of early experiences on the maturation of normal biologic processes one can obviate the need to make a series of essentially arbitrary decisions. More positively, the study of neuroendocrine development might References P. 340-341
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also be expected to generate additional hypotheses regarding the means by which early life experiences influence subsequent psychophysiological processes. Instead of measuring the adrenocortical response to some environmental stimulus or stressor, we were, therefore, led to a study of the effects of early life experiences on the development of the 24-hr rhythm in adrenocortical activity. An outstanding characteristic of normal endocrine function is its daily rhythm. Such circadian rhythms are thought to be endogenous and merely synchronized by environmental cues, the most prepotent of which is the daily light-dark cycle. Nevertheless, these rhythms are not necessarily present at birth. Presumably, the development and maintenance of normal rhythmicity depend upon the functional maturation and integrity of central nervous system areas which are presently unknown or incompletely defined. In the nocturnal rat, maintained under a 12-hr light-dark cycle, there is a precise 24-hr rhythm in adrenocortical activity. Plasma corticosterone levels are at their maximum approximately 2 hr before the 12-hr period of darkness begins and at their nadir approximately 2 hr before the onset of the 12-hr period of light (e.g., Guillemin et al., 1959; Critchlow et al., 1963; Saba et al., 1963; Ader et al., 1967). Infant rats were sampled at these times in the light-dark cycle to determine when, during the course of development, the adrenocortical rhythm could first be observed (Ader, 1969). Experimentally naive animals were reared in modified office cabinets which provided for independent control of light-dark cycles. Under these conditions rhythmicity was first evident at 21 days of age. The difference between values sampled 2 hr before darkness and 2 hr before light gradually increased to the magnitude seen in adult animals. The characteristic sex difference in steroid levels developed sometime after 21 days and was evident in samples obtained when the animals were 30 days old. Although the development of rhythmicity at approximately 3 weeks of age is considerably earlier than had previously been observed (Allen and Kendall, 1967), these observations have been confirmed by Hiroshige and Sat0 (1970a) and by Krieger (1973). In a second study (Ader, 1969), litters of rats born within a 24-hr period were split, randomly reassigned to mothers in litters of 10 animals, and randomly divided into handled, shocked, and control groups. This entire population was housed in an open colony room. Handled animals were removed individually from the nest and simply held in the experimenter’s hand for a period of 3 min daily up to but not including the day of sacrifice. Shocked animals were subjected to 3 min of daily electric shock stimulation (0.1 mA increasing with age to a maximum of 1.0 mA) through the grid floor of a separate chamber. Control animals remained undisturbed. Randomly selected litters of handled, shocked, and control animals were sacrificed at 16, 18, 20, 22, and 25 days of age. Trunk blood was collected and plasma levels of corticosterone were assayed according to the method of Glick et al. (1964) as modified in our laboratory (Friedman et al., 1967). The results of this study are shown in Fig. 1. The unmanipulated control animals did not show any evidence of rhythmicity until 25 days of age. The difference between these data and those of the preceding experiment might be a reflection of the greater
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Fig. 1 . Development of the 24-hr rhythm in adrenocortical activity in handled, shocked, and undisturbed control rats reared under a 12-hr lightdark cycle (from Ader, 1969). Light bars = crest values obtained 2 hr before dark; hatched bars = trough values obtained 2 hr before light; vertical lines = standard error of the mean. Mean values are based on groups of 8-17 animals. (Reprinted by permission of the American Association for the Advancement of Science.)
degree of environmental control afforded by the separate office cabinets used to house the animals in contrast to the housing of these litters in the usual open colony room. In contrast to the control group, handled animals showed a significant difference between the steroid values obtained 2 hr before the onset of darkness and 2 hr before the onset of light at 20 days of age, but there was no significant difference at 22 days of age. At 25 days the rhythm was again evident. The failure to uncover a difference at 22 days appears to be due to the relatively high steroid level observed during the period of darkness. Since relatively innocuous stimulation superimposed upon the trough in the 24-hr adrenal cycle is sufficient to cause a significant elevation in corticosterone level (Ader et al., 1967; Ader and Friedman, 1968), this inconsistency in the data may have been the result of extraneous stimulation. With the single exception of the group of females sampled at 20 days of age, animals that had been subjected to daily electric shock stimulation showed a consistent rhythm in corticosterone concentrations beginning at 16 days of age. Electric shock stimulation experienced during the first 2 weeks of life had, then, accelerated the development of the 24-hr light-synchronized rhythm in adrenocortical activity. The animals that experienced handling or electric shock were stimulated at approximately the same time each day. It was possible, therefore, that such repetitive stimulaReferences p. 340-341
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AGE (IN DAYS]
Fig. 2. Development of the 24-hr rhythm in adrenocortical activity in animals reared under a 12-hr lightdark cycle and subjected to electric shock stimulation at random times on each of the first 14 days of life.
tion could have served as a cue to 24 hr and facilitated the development of rhythmicity by virtue of its significance as a “time giver” rather than as neurogenic stimulation, per se. To eliminate this possibility an additional population of animals was subjected to electric shock stimulation at a different time each day. As can be seen in Fig. 2, a consistent rhythm in steroid levels was again observed beginning at 16 days of age. These data indicated that early life experiences, particularly electric shock stimulation, could influence the development of the 24-hr adrenocortical rhythm, a basic biologic process and a hormonal system with wide ranging influence on behavioral as well as physiologic processes. In order to further define the parameters of stimulation that might be optimal in accelerating this maturational process, the next experiment investigated the effects of different frequencies of stimulation experienced during the first 2 weeks of life on development of the 24-hr light-synchronized rhythm in adrenocortical activity. In order to maintain a constant time for sampling, half the population of pregnant rats were housed under a 12-hr light-dark schedule that was 180” out of phase with the remaining animals. At parturition the litters within a single light-dark schedule were randomly redistributed to the lactating females in litters of 8-10 pups. Four litters each were randomly assigned to groups in which the pups were subjected to a 3-min period of electric shock stimulation every other day, once daily, twice daily, or to a control group which remained unmanipulated. Stimulation was imposed during the first 2 weeks of life. On days 14, 16, 18,21 and 25 two complete litters were decapitated either 2 hr before the lights went off or 2 hr before the lights went on, i.e., at the crest and trough, respectively, of the adult rat’s 24-hr adrenocortical cycle. As before, control animals did not display adrenocortical rhythmicity until 25 days of age. For the animals that were stimulated every other day, the crest values were higher than the trough values at the 0.10 level on days 18 and 21, but did not reach a statistically significant level until 25 days. Animals subjected to a single daily period of stimulation displayed an accelerated development of adrenocortical rhythmicity: there was a consistent difference between crest and trough values beginning at 16 days of age. In animals stimulated twice daily, crest values were significantly higher than trough values beginning at 18 days of age.
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The development of adrenocortical rhythmicity at 25 days in unmanipulated control animals and an accelerated maturation in animals subjected to a single daily period of stimulation replicates our previous data. Beyond this, the findings of this experiment suggest that less frequent stimulation during infancy is less effective in accelerating this developmental process, but such infrequent stimulation does have some facilitating effect. Stimulation twice each day, however, did not further influence the rate of development of the adrenocortical rhythm. The observation that animals stimulated twice daily did not evidence rhythmicity until 18 days (ie.,after the animals stimulated once each day) could suggest that there is a curvilinear function relating amount of stimulation during infancy to developmental processes. This would be consistent with observations of a curvilinear function relating the intensity or frequency of early stimulation to adult emotional reactivity (Ader, 1966; Goldman, 1969). Such a common relationship should not, however, be taken to imply that there is any necessary relationship between adrenocortical rhythmicity and emotional reactivity in the rat. In a further effort to define the conditions under which early life experiences would accelerate maturation of the light-synchronized adrenocortical rhythm, a study was designed to determine if there was a “critical” or sensitive period during which stimulation would be maximally effective in accelerating development. Randomly selected split litters of rats were subjected to 3 min of electric shock stimulation each day during the first week of life, during the second week of life, or during the first and second weeks of life. Control litters remained totally undisturbed. At 16, 18, 20, and 22 days of age randomly selected litters from each group were sacrificed either 2 hr
:1
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Fig. 3. Development of the 24-hr rhythm in adrenocortical activity in rats subjected to electric shock stimulation during different periods of early life. Light bars = crest values; hatched bars = trough values. Mean values are based on groups of 4-10 litters. References p . 340-341
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before the lights went on or 2 hr before the lights went off in the 12-hr light-dark schedule. The results are shown in Fig. 3. Unmanipulated control animals evidenced adrenocortical rhythmicity at 20 days of age which is somewhat earlier than usual for our animals that are maintained in an open colony room. In the animals that experienced electric shock stimulation during the first 2 weeks of life the difference between crest and trough values reached statistically significant levels beginning at 18 days of age, which is somewhat later than had been observed previously. Nonetheless, as before, the animals that had been stimulated showed the expected accelerated maturation of adrenocortical rhythmicity relative to unmanipulated controls. Electric shock stimulation experienced only during the first week of life or only during the second week of life was not sufficient to accelerate the development of rhythmicity; neither of these groups showed evidence of a daily rhythm until 22 days of age. It would appear, then, that stimulation throughout the first 2 weeks of life is necessary for the accelerated development of the 24-hr adrenocortical rhythm and that there is no evidence for a single period within the first 2 weeks of life when environmental stimulation is especially effective in accelerating rhythmicity. The previous experiment indicated that increasing the amount (i.e., the number of periods of daily stimulation) experienced by the animals did not further accelerate rhythmicity or provide for a more delineated rhythm than was obtained from a single period of daily stimulation. In this instance, however, what now appears to have been a relatively moderate amount of stimulation was distributed over the first 2 weeks of life. The possibility remains, therefore, that a major increase in the amount or intensity of stimulation experienced during a shorter period of time could reveal a “critical” period for the effects of stimulation on the maturation of adrenal rhythmicity. It is likely, however, that normal anatomic or functional development within the central nervous system may place a limit on maturation rate. Although the present data provide no evidence for a critical period for the effects of early experience on maturation of the 24-hr adrenocortical rhythm, they have consistently shown that stimulation of the infant rat during the first 2 weeks of life can accelerate the development of this rhythm and, presumably, other rhythmic phenomena. While these studies derived from observations of reduced adrenocortical reactivity in animals stimulated during early life, the present data on accelerated maturation of adrenal function may have no bearing on the mechanisms underlying attenuated adrenocortical reactivity. Several studies have directly or indirectly indicated that different mechanisms appear to be responsible for the ontogenesis of periodicity and the ontogenesis of adrenal responsiveness to environmental or “stressful” stimulation. The means by which early life experiences might hasten the display of rhythmicity remain unknown. There are, however, some parallel data which are of interest and, perhaps, of some import. For example, Hiroshige and Sat0 (1970a, b) have shown that, like corticosterone, the circadian rhythm in hypothalamic corticotropin-releasing factor (CRF) also develops during the third week of life in the rat. While plasma corticosterone levels typically follow changes in CRF activity in the hypothalamus,
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the circadian rhythm in CRF is not controlled by a feedback mechanism. It is evidently under direct control of the central nervous system since the rhythm in CRF activity is present in hypophysectomized as well as in adrenalectomized animals (Hiroshige and Sakakura, 1971; Seiden and Brodish, 1972). Such data suggest that stimulation of the infant rat could be influencing maturation within the central nervous system. Krieger (1973) has shown that a light-dark cycle is necessary for the development and maintenance of normal periodicity in adrenal activity and that the eyes are necessary for the expression of such rhythmicity. Light must, then, be processed by the central nervous system. In this connection, it is of interest to note that Fiske and Leeman (1964) found that neurosecretory material in the supraoptic and paraventricular nuclei of the hypothalamus begins to appear between 14 and 18 days of age which is about the time that the eyes open in the infant rat. By 22 days of age the entire neurosecretory system was functioning actively. As these authors pointed out, “a marked development of the neurosecretory system occurred just as the diurnal corticosteroid rhythm was initiated.” (p. 237). More recently, Campbell and Ramaley (1974) were able to demonstrate a relationship between the development of the 24-hr light-synchronized adrenocortical rhythm and changes in visual input to the hypothalamus. Direct retinohypothalamic projections to the suprachiasmatic nuclei were first observed when the rat was about 17 days of age. Given the temporal relationship between anatomical and functional maturation within the central nervous system and the ontogeny of adrenocortical rhythmicity, it seems possible that the environmental stimulation experienced by the infant rat may be accelerating maturation by accelerating the development of sensory capacity and/or by accelerating anatomical and/or functional development within the central nervous system. There are some data which indicate that early life experiences can influence development of the central nervous system. Using the amount of cholesterol present in whole brain as a reflection of the process of myelination, Levine and Alpert (1959) observed a more rapid development in rats subjected to a handling procedure than in undisturbed controls. Schapiro and Vukovich (1970) studied the development of dendritic spines of cortical pyramidal cells. In the rats subjected to a variety of sensory stimuli several times each day, there was an accelerated development reflected in an increase in the number of dendritic spines observable at 8 days of age and an increase in the number of neurons staining at 8-16 days. It was suggested that the accelerated development of dendritic spines could represent a neuroanatomical basis for the effects of early stimulation upon subsequent behavioral processes. The present data indicate that early life experiences are capable of accelerating the development of adrenocortical rhythmicity. There is no evidence, however, for a critical period for this effect of early stimulation - at least in terms of the brief daily stimulation that has been used thus far. Still, there is accelerated rhythmicity. To the extent that the development and maintenance of the adrenocortical rhythm are a reflection of integrated neuroendocrine function, stimulation during early life would appear to be facilitating the maturation of neuroendocrine mechanisms which, if not directly controlling periodicity, are necessary for the expression of rhythmic processes. References P. 340-341
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SUMMARY
The 24-hr light-synchronized rhythm in plasma levels of corticosterone is first observed in experimentally naive rats between 22 and 25 days of age. Environmental stimulation of the infant rat by handling or, particularly, electric shock stimulation accelerates development of the adrenocortical rhythm. In animals subjected to daily electric shock stimulation rhythmicity is apparent by 16 days of age. Stimulation experienced every other day during the first 2 weeks of life is not quite as effective as daily stimulation, nor does an increase in the frequency of stimulation further accelerate development. Stimulation during the first, second, or first and second weeks of life uncovered no evidence for a “critical” period for the effects of early experiences on the accelerated maturation of adrenocortical rhythmicity. Normal development of the adrenal rhythm is associated with maturational processes within the central nervous system. Although the mechanism remains unknown, it is suggested that early life experiences influence the development of these processes which are necessary for the expression of rhythmic phenomena.
ACKNOWLEDGEMENTS
Preparation of this manuscript and research conducted by the author were supported by U.S.P.H.S. Grant MH-16,741 and a Research Scientist Award (MH-6318) from the National Institute of Mental Health, and a research grant from the Grant Foundation, Inc. REFERENCES ADER,R. (1966) Frequency of stimulation during early life and subsequent emotionality in the rat. Psychol. Rep., 18, 695-701. ADER,R. (1969) Early experiences accelerate maturation of the 24-hour adrenocortical rhythm. Science, 163, 1225-1226. ADER,R. (1970) The effects of early life experiences on developmental processes and susceptibility to disease in animals. In Minnesota Symposium on Child Psychology, J. P. HILL(Ed.), Univ. of Minnesota Press, Minneapolis, Minn., pp. 3-35. S. B. (1968) Plasma corticosterone response to environmental stimulation: ADER,R. AND FRIEDMAN, effects of duration of stimulation and the 24-hour adrenocortical rhythm. Neuroendocrinology, 3, 378-386. ADER,R., FRIEDMAN, S. B. AND GROTA,L. J. (1967) “Emotionality” and adrenal cortical function: effects of strain, test, and the 24-hour corticosterone rhythm. Anim. Behav., 15, 3744. ADER,R. AND GROTA,L. J. (1973) Adrenocortical mediation of the effects of early life experiences. In Drug Effects on Neuroendocrine Regulation, Progress in Brain Research, Vol. 39, E. ZIMMERMANN, W. H. GISPEN,B. H. MARKSAND D. DE WIED(Eds.), Elsevier, Amsterdam, pp. 395405. J. W. (1967) Maturation of the circadian rhythm of plasma corticosterone ALLEN,C. AND KENDALL, in the rat. Endocrinology, 80, 926-930. J. A. (1974) Retinohypothalamic projections: correlations with CAMPBELL, C. B. G. AND RAMALEY, onset of the adrenal rhythm in infant rats. Endocrinology, 94, 1201-1204. CRITCHLOW, V., LIEBELT, R. A,, BAR-SELA, M., MOUNTCASTLE, W. AND LIPSCOMB, H. D. (1963) Sex differences in resting pituitary-adrenal function in the rat. Amer. J. Physiol., 205, 807-815. DENENBERG, V. H. (Ed.) (1972) The Development of Behavior, Sinauer, Stanford, Conn.
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DENENBERG, V. H. AND ZARROW, M. X. (1971) Effects of handling in infancy upon adult behavior and adrenocortical activity: suggestions for a neuroendocrine mechanism. In Eurh Childhood: the Development of Setf-Regulatory Mechanisms, D. H. WALCHER AND D . L. PETERS (Eds.), Academic Press, New York, pp. 39-64. FISKE, V. M. AND LEEMAN, S. (1964) Observations on adrenal rhythmicity and associated phenomena in the rat: effect of light on adrenocortical functions; maturation of the hypothalamic neurosecretory system in relation to adrenal secretion. Ann. N . Y. Acud. Sci., 117, 231-240. FRIEDMAN, S. B. AND ADER,R. (1967) Adrenocortical response to novelty and noxious stimulation. Neuroendocrinology ,2,209-2 12. FRIEDMAN, S. B., ADER, R., GROTA, L. J. AND LARSON, T. (1967) Plasma corticosterone response to parameters of electric shock stimulation in the rat. Psychosom. Med., 29, 323-328. GLICK, D., VON REDLICH, D. AND LEVINE, s.(1964) Fluorometric determination of corticosterone and cortisol in 0.02-0.05 milliliters of plasma or submilligram samples of adrenal tissue. Endocrinology, 74, 653-655. GOLDMAN, P. S. (1969) The relationship between amount of stimulation in infancy and subsequent emotionality. Ann. N . Y. Acud. Sci., 159, 640-650. GLJILLEMIN, R., DEAR, W. E. AND LIEBELT, R. (1959) Nychthermal variations in plasma free corticosteroid levels of the rat. Proc. SOC.exp. Biol. ( N . Y.), 101, 394-395. HIROSHIGE, T. AND SAKAKURA, M. (1971) Circadian rhythm of corticotropin-releasing activity in the hypothalamus of normal and adrenalectomized rats. Neuroendocrinology, 7, 25-36. HIROSHIGE, T. AND SATO,T. (1970a) Circadian rhythm and stress-induced changes in hypothalamic content of corticotropin-releasing activity during postnatal development in the rat. Endocrinology, 86, 1184-1186. HIROSHIGE, T. AND SATO,T. (1970b) Postnatal development of circadian rhythm of corticotropinreleasing activity in the rat hypothalamus. Endocr. jup., 17, 1-6. KRIEGER, D. T. (1973) Effect of ocular enucleation and altered lighting regimens at various ages on the circadian periodicity of plasma corticosteroid levels in the rat. Endocrinology, 93, 1077-1091. LEVINE, S. AND ALPERT, M. (1959) Differential maturation of the central nervous system as a function of early experience. Arch. gen. Psychiut., 1, 403405. LEVINE, S. AND MULLINS, JR., R. J. (1966) Hormonal influence on brain organization in infant rats. Science, 152, 1585-1592. SABA,G. S., SABA,P., CARNICELLI, A. AND M A R E S C OV.~ ,(1963) Diurnal rhythm in the adrenal cortical secretion and in the rate of metabolism of corticosterone in the rat. I. In normal animals. Actu endocr. (Kbh.), 44, 409-412. SCHAPIRO, S. AND VUKOVICH, K. R. (1970) Early experience effects upon cortical dendrites: a proposed model for development. Science, 167, 292-294. SEIDEN, G. AND BRODISH, A. (1972) Persistence of a diurnal rhythm in hypothalamic corticotropinreleasing factor (CRF) in the absence of hormone feedback. Endocrinology, 90, 1401-1403. ZARROW, M. X., DENENBERG, V. H., HALTMEYER, G. C. AND BRUMAGHIN, J. T. (1967) Plasma and adrenal corticosterone levels following exposure of the two-day-old rat to various stressors. Proc. Soc. exp. Biol. (N.Y.), 125, 113-116. ZARROW, M. X., HALTMEYER, G. C., DENENBERG, V. H. AND THATCHER, J. (1966) Response of the infantile rat to stress. Endocrinology, 79, 631-634.
Experimentally imposed stimulation of the infant rat during the first 3 weeks of life is capable of modifying a variety of developmental processes, subsequent behaviors and physiological function, and ultimate susceptibility to experimentally induced organic disease (Ader, 1970; Denenberg, 1972). Based on the consistent finding that infant rats subjected to handling or electric shock stimulation show a reduced adrenocortical response to “stress” experienced later in life, several attempts have been made to relate adult adrenocortical and emotional reactivity to the release of steroids by the neonate in response to the stimulation or “stress” experienced during development (Levine and Mullins, 1966; Denenberg and Zarrow, 1971). A critical review of this literature has been presented in a previous volume in this series (Ader and Grota, 1973). Thus far the effects of early life experiences on subsequent adrenal function have been primarily concerned with adrenocortical reactivity. “Reactivity”, however, is a difficult dimension of adrenal function to define. Several studies (e.g., Zarrow et al., 1966, 1967; Ader et al., 1967; Friedman and Ader, 1967; Ader and Friedman, 1968) have shown that the adrenocortical response to environmental stimulation is determined by a complex interaction among several factors. These include the nature of the stimulus used to elicit an adrenal response, the duration or intensity of that stimulation, the time following stimulation when steroid samples are obtained, and the point in the 24-hr adrenocortical rhythm upon which the stimulation is superimposed. One might also question the criteria defining “reactivity”. Is “reactivity” reflected by the magnitude of the adrenocortical response, the latency of the responses and/or the duration of the elevation in steroid level? All of the above factors are relevant in defining and evaluating adrenocortical reactivity, particularly if one is interested in comparing differentially treated groups of animals. In view of these practical issues, studies of the neuroendocrine mediation of the behavioral and physiological effects of early life experiences become difficult to execute and to interpret. Many of these difficulties may be circumvented by studying the development of normal endocrine function. That is, by analyzing the effects of early experiences on the maturation of normal biologic processes one can obviate the need to make a series of essentially arbitrary decisions. More positively, the study of neuroendocrine development might References P. 340-341
334
R. ADER
also be expected to generate additional hypotheses regarding the means by which early life experiences influence subsequent psychophysiological processes. Instead of measuring the adrenocortical response to some environmental stimulus or stressor, we were, therefore, led to a study of the effects of early life experiences on the development of the 24-hr rhythm in adrenocortical activity. An outstanding characteristic of normal endocrine function is its daily rhythm. Such circadian rhythms are thought to be endogenous and merely synchronized by environmental cues, the most prepotent of which is the daily light-dark cycle. Nevertheless, these rhythms are not necessarily present at birth. Presumably, the development and maintenance of normal rhythmicity depend upon the functional maturation and integrity of central nervous system areas which are presently unknown or incompletely defined. In the nocturnal rat, maintained under a 12-hr light-dark cycle, there is a precise 24-hr rhythm in adrenocortical activity. Plasma corticosterone levels are at their maximum approximately 2 hr before the 12-hr period of darkness begins and at their nadir approximately 2 hr before the onset of the 12-hr period of light (e.g., Guillemin et al., 1959; Critchlow et al., 1963; Saba et al., 1963; Ader et al., 1967). Infant rats were sampled at these times in the light-dark cycle to determine when, during the course of development, the adrenocortical rhythm could first be observed (Ader, 1969). Experimentally naive animals were reared in modified office cabinets which provided for independent control of light-dark cycles. Under these conditions rhythmicity was first evident at 21 days of age. The difference between values sampled 2 hr before darkness and 2 hr before light gradually increased to the magnitude seen in adult animals. The characteristic sex difference in steroid levels developed sometime after 21 days and was evident in samples obtained when the animals were 30 days old. Although the development of rhythmicity at approximately 3 weeks of age is considerably earlier than had previously been observed (Allen and Kendall, 1967), these observations have been confirmed by Hiroshige and Sat0 (1970a) and by Krieger (1973). In a second study (Ader, 1969), litters of rats born within a 24-hr period were split, randomly reassigned to mothers in litters of 10 animals, and randomly divided into handled, shocked, and control groups. This entire population was housed in an open colony room. Handled animals were removed individually from the nest and simply held in the experimenter’s hand for a period of 3 min daily up to but not including the day of sacrifice. Shocked animals were subjected to 3 min of daily electric shock stimulation (0.1 mA increasing with age to a maximum of 1.0 mA) through the grid floor of a separate chamber. Control animals remained undisturbed. Randomly selected litters of handled, shocked, and control animals were sacrificed at 16, 18, 20, 22, and 25 days of age. Trunk blood was collected and plasma levels of corticosterone were assayed according to the method of Glick et al. (1964) as modified in our laboratory (Friedman et al., 1967). The results of this study are shown in Fig. 1. The unmanipulated control animals did not show any evidence of rhythmicity until 25 days of age. The difference between these data and those of the preceding experiment might be a reflection of the greater
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Fig. 1 . Development of the 24-hr rhythm in adrenocortical activity in handled, shocked, and undisturbed control rats reared under a 12-hr lightdark cycle (from Ader, 1969). Light bars = crest values obtained 2 hr before dark; hatched bars = trough values obtained 2 hr before light; vertical lines = standard error of the mean. Mean values are based on groups of 8-17 animals. (Reprinted by permission of the American Association for the Advancement of Science.)
degree of environmental control afforded by the separate office cabinets used to house the animals in contrast to the housing of these litters in the usual open colony room. In contrast to the control group, handled animals showed a significant difference between the steroid values obtained 2 hr before the onset of darkness and 2 hr before the onset of light at 20 days of age, but there was no significant difference at 22 days of age. At 25 days the rhythm was again evident. The failure to uncover a difference at 22 days appears to be due to the relatively high steroid level observed during the period of darkness. Since relatively innocuous stimulation superimposed upon the trough in the 24-hr adrenal cycle is sufficient to cause a significant elevation in corticosterone level (Ader et al., 1967; Ader and Friedman, 1968), this inconsistency in the data may have been the result of extraneous stimulation. With the single exception of the group of females sampled at 20 days of age, animals that had been subjected to daily electric shock stimulation showed a consistent rhythm in corticosterone concentrations beginning at 16 days of age. Electric shock stimulation experienced during the first 2 weeks of life had, then, accelerated the development of the 24-hr light-synchronized rhythm in adrenocortical activity. The animals that experienced handling or electric shock were stimulated at approximately the same time each day. It was possible, therefore, that such repetitive stimulaReferences p. 340-341
336
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AGE (IN DAYS]
Fig. 2. Development of the 24-hr rhythm in adrenocortical activity in animals reared under a 12-hr lightdark cycle and subjected to electric shock stimulation at random times on each of the first 14 days of life.
tion could have served as a cue to 24 hr and facilitated the development of rhythmicity by virtue of its significance as a “time giver” rather than as neurogenic stimulation, per se. To eliminate this possibility an additional population of animals was subjected to electric shock stimulation at a different time each day. As can be seen in Fig. 2, a consistent rhythm in steroid levels was again observed beginning at 16 days of age. These data indicated that early life experiences, particularly electric shock stimulation, could influence the development of the 24-hr adrenocortical rhythm, a basic biologic process and a hormonal system with wide ranging influence on behavioral as well as physiologic processes. In order to further define the parameters of stimulation that might be optimal in accelerating this maturational process, the next experiment investigated the effects of different frequencies of stimulation experienced during the first 2 weeks of life on development of the 24-hr light-synchronized rhythm in adrenocortical activity. In order to maintain a constant time for sampling, half the population of pregnant rats were housed under a 12-hr light-dark schedule that was 180” out of phase with the remaining animals. At parturition the litters within a single light-dark schedule were randomly redistributed to the lactating females in litters of 8-10 pups. Four litters each were randomly assigned to groups in which the pups were subjected to a 3-min period of electric shock stimulation every other day, once daily, twice daily, or to a control group which remained unmanipulated. Stimulation was imposed during the first 2 weeks of life. On days 14, 16, 18,21 and 25 two complete litters were decapitated either 2 hr before the lights went off or 2 hr before the lights went on, i.e., at the crest and trough, respectively, of the adult rat’s 24-hr adrenocortical cycle. As before, control animals did not display adrenocortical rhythmicity until 25 days of age. For the animals that were stimulated every other day, the crest values were higher than the trough values at the 0.10 level on days 18 and 21, but did not reach a statistically significant level until 25 days. Animals subjected to a single daily period of stimulation displayed an accelerated development of adrenocortical rhythmicity: there was a consistent difference between crest and trough values beginning at 16 days of age. In animals stimulated twice daily, crest values were significantly higher than trough values beginning at 18 days of age.
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The development of adrenocortical rhythmicity at 25 days in unmanipulated control animals and an accelerated maturation in animals subjected to a single daily period of stimulation replicates our previous data. Beyond this, the findings of this experiment suggest that less frequent stimulation during infancy is less effective in accelerating this developmental process, but such infrequent stimulation does have some facilitating effect. Stimulation twice each day, however, did not further influence the rate of development of the adrenocortical rhythm. The observation that animals stimulated twice daily did not evidence rhythmicity until 18 days (ie.,after the animals stimulated once each day) could suggest that there is a curvilinear function relating amount of stimulation during infancy to developmental processes. This would be consistent with observations of a curvilinear function relating the intensity or frequency of early stimulation to adult emotional reactivity (Ader, 1966; Goldman, 1969). Such a common relationship should not, however, be taken to imply that there is any necessary relationship between adrenocortical rhythmicity and emotional reactivity in the rat. In a further effort to define the conditions under which early life experiences would accelerate maturation of the light-synchronized adrenocortical rhythm, a study was designed to determine if there was a “critical” or sensitive period during which stimulation would be maximally effective in accelerating development. Randomly selected split litters of rats were subjected to 3 min of electric shock stimulation each day during the first week of life, during the second week of life, or during the first and second weeks of life. Control litters remained totally undisturbed. At 16, 18, 20, and 22 days of age randomly selected litters from each group were sacrificed either 2 hr
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Fig. 3. Development of the 24-hr rhythm in adrenocortical activity in rats subjected to electric shock stimulation during different periods of early life. Light bars = crest values; hatched bars = trough values. Mean values are based on groups of 4-10 litters. References p . 340-341
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before the lights went on or 2 hr before the lights went off in the 12-hr light-dark schedule. The results are shown in Fig. 3. Unmanipulated control animals evidenced adrenocortical rhythmicity at 20 days of age which is somewhat earlier than usual for our animals that are maintained in an open colony room. In the animals that experienced electric shock stimulation during the first 2 weeks of life the difference between crest and trough values reached statistically significant levels beginning at 18 days of age, which is somewhat later than had been observed previously. Nonetheless, as before, the animals that had been stimulated showed the expected accelerated maturation of adrenocortical rhythmicity relative to unmanipulated controls. Electric shock stimulation experienced only during the first week of life or only during the second week of life was not sufficient to accelerate the development of rhythmicity; neither of these groups showed evidence of a daily rhythm until 22 days of age. It would appear, then, that stimulation throughout the first 2 weeks of life is necessary for the accelerated development of the 24-hr adrenocortical rhythm and that there is no evidence for a single period within the first 2 weeks of life when environmental stimulation is especially effective in accelerating rhythmicity. The previous experiment indicated that increasing the amount (i.e., the number of periods of daily stimulation) experienced by the animals did not further accelerate rhythmicity or provide for a more delineated rhythm than was obtained from a single period of daily stimulation. In this instance, however, what now appears to have been a relatively moderate amount of stimulation was distributed over the first 2 weeks of life. The possibility remains, therefore, that a major increase in the amount or intensity of stimulation experienced during a shorter period of time could reveal a “critical” period for the effects of stimulation on the maturation of adrenal rhythmicity. It is likely, however, that normal anatomic or functional development within the central nervous system may place a limit on maturation rate. Although the present data provide no evidence for a critical period for the effects of early experience on maturation of the 24-hr adrenocortical rhythm, they have consistently shown that stimulation of the infant rat during the first 2 weeks of life can accelerate the development of this rhythm and, presumably, other rhythmic phenomena. While these studies derived from observations of reduced adrenocortical reactivity in animals stimulated during early life, the present data on accelerated maturation of adrenal function may have no bearing on the mechanisms underlying attenuated adrenocortical reactivity. Several studies have directly or indirectly indicated that different mechanisms appear to be responsible for the ontogenesis of periodicity and the ontogenesis of adrenal responsiveness to environmental or “stressful” stimulation. The means by which early life experiences might hasten the display of rhythmicity remain unknown. There are, however, some parallel data which are of interest and, perhaps, of some import. For example, Hiroshige and Sat0 (1970a, b) have shown that, like corticosterone, the circadian rhythm in hypothalamic corticotropin-releasing factor (CRF) also develops during the third week of life in the rat. While plasma corticosterone levels typically follow changes in CRF activity in the hypothalamus,
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the circadian rhythm in CRF is not controlled by a feedback mechanism. It is evidently under direct control of the central nervous system since the rhythm in CRF activity is present in hypophysectomized as well as in adrenalectomized animals (Hiroshige and Sakakura, 1971; Seiden and Brodish, 1972). Such data suggest that stimulation of the infant rat could be influencing maturation within the central nervous system. Krieger (1973) has shown that a light-dark cycle is necessary for the development and maintenance of normal periodicity in adrenal activity and that the eyes are necessary for the expression of such rhythmicity. Light must, then, be processed by the central nervous system. In this connection, it is of interest to note that Fiske and Leeman (1964) found that neurosecretory material in the supraoptic and paraventricular nuclei of the hypothalamus begins to appear between 14 and 18 days of age which is about the time that the eyes open in the infant rat. By 22 days of age the entire neurosecretory system was functioning actively. As these authors pointed out, “a marked development of the neurosecretory system occurred just as the diurnal corticosteroid rhythm was initiated.” (p. 237). More recently, Campbell and Ramaley (1974) were able to demonstrate a relationship between the development of the 24-hr light-synchronized adrenocortical rhythm and changes in visual input to the hypothalamus. Direct retinohypothalamic projections to the suprachiasmatic nuclei were first observed when the rat was about 17 days of age. Given the temporal relationship between anatomical and functional maturation within the central nervous system and the ontogeny of adrenocortical rhythmicity, it seems possible that the environmental stimulation experienced by the infant rat may be accelerating maturation by accelerating the development of sensory capacity and/or by accelerating anatomical and/or functional development within the central nervous system. There are some data which indicate that early life experiences can influence development of the central nervous system. Using the amount of cholesterol present in whole brain as a reflection of the process of myelination, Levine and Alpert (1959) observed a more rapid development in rats subjected to a handling procedure than in undisturbed controls. Schapiro and Vukovich (1970) studied the development of dendritic spines of cortical pyramidal cells. In the rats subjected to a variety of sensory stimuli several times each day, there was an accelerated development reflected in an increase in the number of dendritic spines observable at 8 days of age and an increase in the number of neurons staining at 8-16 days. It was suggested that the accelerated development of dendritic spines could represent a neuroanatomical basis for the effects of early stimulation upon subsequent behavioral processes. The present data indicate that early life experiences are capable of accelerating the development of adrenocortical rhythmicity. There is no evidence, however, for a critical period for this effect of early stimulation - at least in terms of the brief daily stimulation that has been used thus far. Still, there is accelerated rhythmicity. To the extent that the development and maintenance of the adrenocortical rhythm are a reflection of integrated neuroendocrine function, stimulation during early life would appear to be facilitating the maturation of neuroendocrine mechanisms which, if not directly controlling periodicity, are necessary for the expression of rhythmic processes. References P. 340-341
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SUMMARY
The 24-hr light-synchronized rhythm in plasma levels of corticosterone is first observed in experimentally naive rats between 22 and 25 days of age. Environmental stimulation of the infant rat by handling or, particularly, electric shock stimulation accelerates development of the adrenocortical rhythm. In animals subjected to daily electric shock stimulation rhythmicity is apparent by 16 days of age. Stimulation experienced every other day during the first 2 weeks of life is not quite as effective as daily stimulation, nor does an increase in the frequency of stimulation further accelerate development. Stimulation during the first, second, or first and second weeks of life uncovered no evidence for a “critical” period for the effects of early experiences on the accelerated maturation of adrenocortical rhythmicity. Normal development of the adrenal rhythm is associated with maturational processes within the central nervous system. Although the mechanism remains unknown, it is suggested that early life experiences influence the development of these processes which are necessary for the expression of rhythmic phenomena.
ACKNOWLEDGEMENTS
Preparation of this manuscript and research conducted by the author were supported by U.S.P.H.S. Grant MH-16,741 and a Research Scientist Award (MH-6318) from the National Institute of Mental Health, and a research grant from the Grant Foundation, Inc. REFERENCES ADER,R. (1966) Frequency of stimulation during early life and subsequent emotionality in the rat. Psychol. Rep., 18, 695-701. ADER,R. (1969) Early experiences accelerate maturation of the 24-hour adrenocortical rhythm. Science, 163, 1225-1226. ADER,R. (1970) The effects of early life experiences on developmental processes and susceptibility to disease in animals. In Minnesota Symposium on Child Psychology, J. P. HILL(Ed.), Univ. of Minnesota Press, Minneapolis, Minn., pp. 3-35. S. B. (1968) Plasma corticosterone response to environmental stimulation: ADER,R. AND FRIEDMAN, effects of duration of stimulation and the 24-hour adrenocortical rhythm. Neuroendocrinology, 3, 378-386. ADER,R., FRIEDMAN, S. B. AND GROTA,L. J. (1967) “Emotionality” and adrenal cortical function: effects of strain, test, and the 24-hour corticosterone rhythm. Anim. Behav., 15, 3744. ADER,R. AND GROTA,L. J. (1973) Adrenocortical mediation of the effects of early life experiences. In Drug Effects on Neuroendocrine Regulation, Progress in Brain Research, Vol. 39, E. ZIMMERMANN, W. H. GISPEN,B. H. MARKSAND D. DE WIED(Eds.), Elsevier, Amsterdam, pp. 395405. J. W. (1967) Maturation of the circadian rhythm of plasma corticosterone ALLEN,C. AND KENDALL, in the rat. Endocrinology, 80, 926-930. J. A. (1974) Retinohypothalamic projections: correlations with CAMPBELL, C. B. G. AND RAMALEY, onset of the adrenal rhythm in infant rats. Endocrinology, 94, 1201-1204. CRITCHLOW, V., LIEBELT, R. A,, BAR-SELA, M., MOUNTCASTLE, W. AND LIPSCOMB, H. D. (1963) Sex differences in resting pituitary-adrenal function in the rat. Amer. J. Physiol., 205, 807-815. DENENBERG, V. H. (Ed.) (1972) The Development of Behavior, Sinauer, Stanford, Conn.
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