Multifactorial regulation of the hypothalamic-pituitary-adrenal axis during development

NeuroscienceandBiobehavioralReviews,Vol. 16, pp. 553-568, 1992

0149-7634/92$5.00 + .00 Copyrighto 1992PergamonPress Ltd.

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Multifactorial Regulation of the Hypothalamic-Pituitary-Adrenal Axis During Development PATRICIA ROSENFELD, DEBORAH SUCHECKI AND SEYMOUR LEVINE l

Department o f Psychiatry and Behavioral Sciences, Stanford University School o f Medicine, Stanford University, Stanford, CA 94305 Received 12 J u n e 1992 ROSENFELD, P., D. SUCHECKI AND S. LEVINE. Multifactorial regulation of the hypothalamic-pituitary-adrenal axis during development. NEUROSCI BIOBEHAV REV 16(4) 553-568, 1992.-The hypothalamic-pituitary-adrenal system shows an overall diminished responsiveness throughout ontogeny. Thus, during this period, the sensitivity of the adrenal gland to ACTH is markedly reduced. Furthermore, basal and stress-inducedconcentrations of corticosterone (CORT), ACTH and hypothalamic secretagogues remain at very low levels. Both structural immaturity and active inhibitory processes appear to underlie this overall hyporesponsiveness. The available data indicate that the characteristic developmental pattern of the HPA system results from multiple regulatory factors acting in conjunction at various levels of the axis. The primary ratelimiting steps, however, are probably at the brain and adrenal levels. The ultimate "goal" appears to be to keep CORT levels within the narrow range of concentrations required for normal development. Hypothalamic-pituitary-adrenal a x i s

Stresshyporesponsive period

Development

Maternal deprivation

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mechanisms that appear to operate during this period serves as teleological proof that this delicate balance is crucial for normal development. In the following pages we will briefly review the available literature on this topic. Particular emphasis shall be placed on those factors that appear to directly regulate the output of the HPA system during postnatal development in the rat.

THE infant rat shows a markedly diminished adrenocortical response to stress. This was first reported almost 50 years ago (65); S. Schapiro coined the phrase "stress nonresponsive period" (SNRP) to refer to this phenomenon (141). In the ensuing years, a vast number of studies have both corroborated and expanded on these early observations. The availability of more sensitive assays revealed that the infant does show a small adrenocortical response to specific stimuli (6,145,179,180,184,186). This has lead to a change in nomenclature: The term "stress hyporesponsive period" (SHRP) has replaced SNRP to emphasize this phenomenon (145). Furthermore, we now know that this diminished responsiveness is not limited to adrenal output; rather, it reflects an overall suppression of the infant's hypothalamic-pituitary-adrenal (HPA) system. In addition, we know that under certain conditions this suppression can at least partially be overridden [for reviews see (37,130,185)]. We do not as yet have, however, a definitive answer regarding the underlying causative mechanisms, nor do we have a clear picture of the regulation of the HPA axis during this period. Answers to the above questions are important for a number of reasons. On the one hand, low levels of glucocorticoids (GCs) are essential for normal development [for reviews see (7,38,95)]. Excessive GCs, in contrast, have widespread deleterious effects [for review see (12)]. The array of regulatory

THE SHRP: IMMATURITYOR INHIBITION? From approximately postnatal day (pnd) 4 to pnd 14, most commonly used stressors fail to elicit an adrenocortical response in the infant rat, or do so only minimally [for reviews see (37,130)]. A closer inspection reveals that this diminished responsiveness characterizes all of the components of the HPA axis. Thus, not only does the animal secrete minimal amounts of corticosterone (CORT, the main GC in the rat), it has in addition low levels of adrenocorticotropin (ACTH) and hypothalamic secretagogues [e.g., corticotropin-releasing-factor (CRF) and arginine-vasopressin (AVP)] (48,87,123, 178,190). Given the extent of postnatal development that takes place in the rat brain, it is also highly likely that neural connections to the hypothalamus differ from those of the adult. Furthermore, the adrenal gland is markedly hyporesponsive to its tropic hormone (51,88,117). Finally, there are profound

To whom requests for reprints should be addressed. 553

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ROSENFELD, SUCHECKI AND LEVINE

changes in CORT transport and metabolism: Plasma and pituitary levels of corticosterone-binding-globulin (CBG) decrease dramatically in the first few postnatal days, and remain at or below detection levels throughout the SHRP; the half-life of CORT decreases slowly with age (57,127,128,142,170). The final outcome of this set of events is a period, which covers most of the animal's infancy, during which plasma CORT remains at low, relatively unperturbable levels. Is this primarily due to immaturity of the underlying structures, or are inhibitory processes taking place? The correct answer is, probably, both of the above. Thus, the perinatal rat is capable of a vigorous adrenocortical response, but only following certain stimuli (6,178,190). Furthermore, the foetal rat is capable of secreting large amounts of ACTH and CORT (13,23,24, 40,98); adrenal hyporesponsivity to ACTH does not develop until pnd 4, approximately (88,190,192). These data suggest that the lack of a response during the SHRP is not merely due to immaturity of the central components of the H P A axis. INTRINSIC VERSUS EXTRINSIC REGULATORY FACTORS Most research has focussed on intrinsic factors regulating the H P A system during ontogeny. Recently, however, we and others have shown that extrinsic factors must also be taken into account when attempting to explain H P A regulation during this period. Specifically, maternal factors appear to exert a strong inhibitory effect on the infant's H P A system. This can be demonstrated by removing the source of regulation, i.e., the mother. Following prolonged (e.g., 24 h) maternal deprivation, the infant shows a marked increase in adrenal responsiveness to ACTH, and, at certain ages, in basal and stress-induced CORT and ACTH secretion (26,78,89,117, 154,156). The changes in CORT secretion can be mostly reversed by feeding the pup during the deprivation period (116). Preliminary data, however, indicate that changes in ACTH secretion are not due to lack of food, a n d / o r loss of body temperature in the deprived infants [unpublished data, (156)]. The effects of short periods of maternal deprivation on CORT secretion do not have a cumulative effect; maternal reunion may be sufficient to "reset" the system at control levels following short periods of deprivation (121). Finally, there appears to be a critical length of deprivation (somewhere between 8 and 24 h) beyond which there are persistent (i.e., at least 4 days following maternal reunion) changes in adrenocortical responsivity, though not in basal CORT secretion. These data suggest that basal and stress-induced CORT secretion in the infant rat are sensitive to different aspects of maternal regulation (121). The available data are insufficient to determine whether the maternal inhibitory effect is mediated at the pituitary and/or brain level. Given the current state of knowledge, however, a central locus of action seems more likely. THE NEGATIVE FEEDBACK HYPOTHESIS If the HPA system can potentially respond during the proposed refractory period, then why does it not do so? A number of theories have been proposed to attempt to explain this question. Of these, perhaps the one that has achieved most attention is the so-called "negative-feedback hypothesis." First outlined by Schapiro (140), and later expanded to include more recent data (128,130,179) the hypothesis in its current form rests on three basic tenets: (a) GC receptors are present in the pituitary in adult-like concentrations already at the time of birth, and these concentrations remain constant throughout development (102,127), (b) Plasma CBG levels follow a Ushaped curve similar to that shown by CORT during the

SHRP (57,170), and (c) CBG-like molecules have been found within the pituitary corticotrophs, in concentrations that parallel those found in plasma, both in the adult and infant organism (36,128). GC receptors in the pituitary (specifically, the Type 2 or GR receptors) have been postulated to mediate the negative feedback effects of GCs on their own secretion [for reviews see (30,35)]. CORT has a far higher affinity for GRs than for CBG (183). Nevertheless, high concentrations of CBG in the pituitary may result in a reduced negative feedback signal at this level by competing with specific GC receptors for CORT. The consequences of low CBG levels during the SHRP would be two-fold. First, most of the CORT present in plasma would be in the free (biologically active) form. Thus, even though total (i.e., free + bound) concentrations of CORT remain low during the SHRP, the biologically active fraction would be relatively large. Second, the decreased presence of CBG in the corticotrophs would allow a greater percentage of CORT to bind to the GRs. The combination of high concentrations of free CORT and pituitary GRs, and low concentrations of pituitary CBG, would lead to an enhanced negative feedback signal at the level of the pituitary. This inhibitory signal would be strong enough to largely prevent any stress signal from breaking through (128,130,179). One way of testing this hypothesis would be to remove the source of endogenous CORT [e.g., via adrenalectomy (ADX)] and replace with exogenous GCs. If negative feedback were indeed enhanced during this period, then, as compared to the adult, in the neonate (a) ADX should lead to a larger increase in ACTH secretion, and (b) ACTH secretion should be inhibited by smaller doses of GCs. The available data indicate that both basal and stressinduced levels of ACTH in ADX infants are significantly higher than in their non-ADX counterparts (54,177,179,186). In addition, younger animals require lower doses of CORT than older animals in order to suppress a urethane- or CRF-induced ACTH response (in vivo and in vitro, respectively) (179). Finally, in ADX animals nuclear uptake of (3H)CORT in the pituitary is significantly higher in neonates than at later ages (127). The abovementioned studies would seem to support the enhanced feedback hypothesis. However, a closer look at the data reveals a number of inconsistencies a n d / o r unresolved issues that raise questions concerning the validity of this hypothesis. The effects of maternal deprivation, for example, are hard to reconcile with the notion of an enhanced negative feedback. Were the infant to have an augmented feedback system, then the deprivation-induced increases in basal CORT levels should lead to a greater feedback signal and, consequently, to a decrease in the responsiveness of the system. Furthermore, the "turning off" of ACTH secretion should be more efficient in the infant than in the adult. In fact, exactly the opposite occurs: maternally-deprived animals, despite elevated basal CORT levels, are hyper, rather than hyporesponsive (156). Moreover, the ACTH response in deprived animals persists far longer than in the adult (156). The precise mechanism through which these maternal signals are transduced remains to be determined (see later sections for further discussion). It should be emphasized that maternal inhibition cannot, on its own, account for the hyporesponsiveness characteristic of the SHRP. Thus, both basal and stress-induced CORT levels in maternally-deprived pups show an ontogenetic U-shaped curve that parallels, albeit at a higher level, that found in non-deprived infants (26,89,117,154,178,190). This suggests that other inhibitory mechanisms are operating at this time.

H P A AXIS AND DEVELOPMENT In summary, there appears to be a multifactorial regulation of the H P A system during the SHRP. This regulation probably involves both extrinsic and intrinsic factors, operating at various levels of the axis. In the following sections we shall attempt to give an overview of what these factors might be, and how they may function. For purposes of exposition we shall arbitrarily divide the axis into its peripheral and central components. ADRENAL LEVEL Metabolism One of the salient features of the SHRP is the U-shaped ontogenetic curve in basal concentrations of plasma CORT (145,178,190). This pattern could, in theory, be accounted for solely by post-adrenal mechanisms. There are data that indicate this might indeed be the case. Thus, infant (i.e., pnd 12, 16, and 20) rats, adrenalectomized and subsequently provided with a constant dose of CORT via subcutaneous pellets, show an age-dependent increase in plasma CORT levels. This increase is similar to that found in age-matched, shamoperated littermates (146). These results indicate there is an ontogenetic decrease in plasma clearance of CORT, most likely due to a decrease in the apparent volume of distribution of the hormone. This latter decrease would be a function of the concurrent increase in plasma CBG levels: CORT binding to CBG markedly reduces tissue uptake of the hormone (146). As referred to previously, CBG levels show a U-shaped developmental pattern remarkably similar to that of circulating basal CORT concentrations (57,170). The above data suggest the U-shaped curve in basal plasma CORT concentrations reflects changes in post-adrenal factors, rather than in CORT synthesis. However, 8-, 12- and 16-dayold pups that have been maternally-deprived for 24 h show a significant increase in basal levels of plasma CORT (89,117). Adrenal CORT concentrations, which presumably are not affected by changes in clearance, follow a very similar pattern (Fig. l). Furthermore, CBG levels, although slightly higher in pnd 10 24-h deprived animals, are actually substantially lower in deprived 16-day-old pups (Fig. 2). Maternal factors thus appear to inhibit basal levels of CORT synthesis and secretion during (and beyond) the SHRP. Adrenal Responsiveness CBG may account for part of the ontogenetic changes in basal plasma CORT levels. However, it cannot explain the profound hyporesponsiveness to stress a n d / o r ACTH that typifies the SHRP: CBG levels do not show a fast response to stress (Fig. 2). As mentioned earlier, during the SHRP the adrenal is refractory to ACTH, in that even large doses of ACTH fail to elicit an adult-like CORT response (26,89, 116,117,154). This refractoriness has been postulated to reflect decreased exposure of the adrenal gland to ACTH during this period (51). In the adult animal, in addition to its acute stimulatory effect on steroidogenesis, ACTH has a t r o p h i c action on its target gland [(106,147) for reviews see (111,149)]. Many of these trophic effects (e.g., ACTH receptor upregulation, maturation of the adenylate cyclase system) are present already in the fetal adrenal (25,39,41,42,125). In addition, ACTH induces the transformation of undifferentiated adrenal cells into functional fasciculata cells, at least in vitro (91). The adrenal hyporesponsiveness during the SHRP may therefore be a consequence of constant low ACTH levels. Maternal deprivation leads to a pronounced increase in

555 adrenal responsiveness to ACTH (26,89,117,154, Fig. 3). Deprivation might thus be hypothesized to result in an increase in ACTH secretion, which would lead in turn (through its trophic actions) to an increase in adrenal responsivity to ACTH. Were this the only mechanism at work, however, we would expect deprived animals at all ages to show higher base levels of CORT. We have found that, although deprivation does lead to an enhanced CORT response to exogenous ACTH at all ages, basal levels are not significantly elevated in younger (i.e., pnd 4) pups (89,117). The existence of additional regulatory mechanisms is supported by a variety of data. Thus, studies carried out in the fetal lamb (equivalent data for the rat are not available), indicate that ACTH only accounts for part of the developmental increase in steroidogenic capacity (at least in vitro) (41). Both inhibitory and stimulatory signals appear to affect adrenocortical cell differentiation and function. The former include, for example, an extra-pituitary factor postulated to inhibit ACTH receptor maturation (125). Among the latter, IGF-II and (possibly) certain pro-opiomelano cortin (POMC)-derived peptides have been shown to have growth-promoting effects [(44,173) for review see (172)]. The effects of maternal deprivation might therefore be due to changes in these signals, rather than being a secondary effect of increased ACTH secretion. In this regard, it is interesting to note that feeding completely suppresses the increases in basal CORT secretion that follow maternal deprivation, and largely reverses stressinduced increases in these animals (116). Feeding does not, however, affect ACTH secretion, suggesting that factors other than ACTH modify adrenal sensitivity (unpublished data). Throughout the SHRP, therefore, the absence of CBG combined with a decrease in adrenal steroidogenic capacity and sensitivity to ACTH, result in low basal levels of (both total and free) circulating CORT. More important, perhaps, is the fact that due to the adrenal hyporesponsiveness these concentrations remain fairly unperturbable. In other words, during this period the infant shows a minimal CORT response to stress, whether or not there is a response (i.e., CRF and/or ACTH secretion) at higher levels of the axis. PITUITARY LEVEL Regardless of whether or not the adrenal is hyporesponsive, however, it is clear that there will be no CORT response unless ACTH is first released from the pituitary. In the following sections we shall examine the pituitary response to both stress and direct stimulation by hypothalamic secretagogues, and discuss the possible factors regulating pituitary output during this period. Pituitary Response to Stress A review of the available data indicates that the neonatal rat can secrete ACTH in response to certain types of stressors. This response is both stimulus-specific and age-dependent. Thus, for example, severe immune challenges (e.g., injection of a LD90 dose of bacterial endotoxin) have been shown to elicit a relatively large increase in plasma ACTH ( - 3 0 0 pg/ml) in the l-day-old pup. In this case, adult-like magnitudes ( - 4 5 0 pg/ml) are already observed at pnd 5 (190). Milder immune challenges appear to result in proportionally smaller ACTH responses: Peak values of - 2 5 0 pg/ml have been reported to occur following histamine injection at pnd 10 (data are not available at other ages) (180). Glucoprivation [induced by insulin or 2-deoxiglucose (2DG) injection] is also capable of inducing a significant plasma

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FIG. 1. Adrenal corticosterone concentration (#g/dl/mg adrenal) in rat pups at different ages. Pups were either nondeprived (NDEP) or maternally-deprived (DEP) for 24 h. During the deprivation period pups were kept at 30-33°C. At the end of the deprivation period (or at the corresponding timepoint for NDEP pups) 1 male and 1 female

from each litter were sacrificed immediately and their adrenals excised (time 0). The remaining pups were injected IP with 1 IU/100 gbw of ACTH~.24and sacrificed 15, 30, or 60 rain later. The adrenals were excised, cleaned, weighed, and homogenized in ethanol to a 1/500 final solution. The solution was centrigued for 20 rain at 2,000 rpm at 2°C; the supernatant was extracted and stored at -20°C until subsequent radioimmunoassay for CORT (117). At each age, data were analyzed by means of a 2 (Conditions: NDEP, DEP) x 4 (Time-Points: 0, 15, 30, 60 min) ANOVA, since sex was not a significant variable. Values correspond to the mean + SEM. •p < 0.05, compared to NDEP pups; l"p < 0.05, compared to time 0.

ACTH response at pnd 11-12 (other ages have not been tested). This response, however, is less than half that found in similarly-treated adults (186). Hypoxia, yet another "physiological" stressor, only stimulates A C T H secretion as from approximately pnd 14 on; the magnitude of the response increases with age (178). Pups have also been shown to respond to other stimuli that may act directly at the hypothalamic level. Ether, a stressor widely used in adult testing, is only moderately effective during early development. Thus, pups do not respond (or do so only minimally) until pnd 10, approximately. From this age until pnd 21 the magnitude of the response does not vary ( - 350 pg/ml); once again, this is about half the adult value. The onset of the response, however, does not seem to differ from that of adults: maximum levels are reached within 5 min, approximately (50,178,180). Ten- and 18-day-old pups also show a similar (albeit larger, - 5 0 0 pg/ml) response to urethane, an extremely potent stimulus (179). All of the above test stimuli have in common that they do not require collative processing in order to activate the H P A axis [for review see (56)]. Less is known about the ontogeny

of the A C T H response to stimuli that do require collative processing. Electric shock does not cause a significant rise in plasma A C T H until close to weaning time (pnd 18). Even then, the response is minimal compared to that of the adult ( - 2 5 0 versus - 1900 pg/ml) (179). There are few data available on the A C T H response to milder psychological stressors, such as saline injection or exposure to novelty. In those cases in which values following saline injection have been reported, the results suggest there is virtually no response until the pups are around 21 days of age (179,180,184). This, however, may reflect an experimental artifact: In all of these studies the saline injection was used as a control for other more potent procedures (e.g., CRF or histamine administration). Thus, small differences following saline injection would probably be statistically undetectable. Recent data from our laboratory, in fact, indicate that small (albeit significant) responses to saline injection can be detected already in the 12-day-old pup (156). The response to novelty, however, seems to develop later in ontogeny. The data reviewed in the preceding paragraphs, although fragmentary, allow certain generalizations to be made. First,

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adult) (133,134). The diminished A C T H output, therefore, m i g h t be postulated to reflect a deficiency in the proteolytic processing m a c h i n e r y in the neonate. T h e r e are data, however, that render this hypothesis implausible. Thus, a n t e r i o r pituitary content o f P O M C increases 5-fold f r o m b i r t h to 4 weeks, and a n o t h e r 3-fold by a d u l t h o o d (133); total A C T H content is also very low in the n e o n a t e (less t h a n 10% t h a t o f the adult animal) (178,186). This does not,

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FIG. 2. Plasma CBG levels ~ g / d l ) in 10- and 16-day-old rat pups. Pups were either nondeprived (NDEP) or maternally-deprived (DEP) for 24 h. During deprivation period pups were kept at 30-33°C. At the end of the deprivation period (or at the corresponding timepoint for NDEP pups) 2 males and 2 females from each litter were blood sampled immediately (BASAL). The remaining pups were injected IP with 0.9% NaC1 (0.1 ml/10 gbw) and sacrificed 30 rain later (SALINE). Serum was centrifuged for 20 min at 2000 rpm at 2°C; the supernatant was extracted and stored at - 2 0 ° C until subsequent binding assay for CBG. At each age, data were analyzed by means of a 2 (Conditions: NDEP, DEP) x 2 (Treatments: basal, saline) ANOVA, since sex was not a significant variable. No differences due to treatment were observed. Values correspond to the mean ± SEM. *p < 0.05; tP < 0.01, compared to NDEP pups.

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the capacity to secrete A C T H in response to certain stimuli is present t h r o u g h o u t d e v e l o p m e n t . Second, the response to different stimuli show m a r k e d l y different ontogenetic patterns. Third, with rare exceptions (e.g., bacterial endotoxin), the m a g n i t u d e o f the A C T H response in the p u p is m a r k e d l y smaller t h a n t h a t f o u n d in the adult a n i m a l following a similar challenge. F o u r t h , w h e n a response does occur, its rise time does not a p p e a r to c h a n g e with age (180). During the S H R P , therefore, there is a sharply diminished pituitary o u t p u t in response to m o s t stressors. This decrease m a y be due to a variety o f causes. These can, however, be b r o k e n d o w n into three m a i n categories: (a) a n inability to produce A C T H [e.g., decreased proteolytic cleavage o f the P O M C molecule], (b) a n excess o f i n h i b i t o r y inputs (e.g., negative feedback), a n d (c) a lack o f stimulatory inputs (e.g., insensitivity to, or decreased availability of, h y p o t h a l a m i c releasing h o r m o n e s ) . In the following sections we shall examine these alternatives.

A C T H Synthesis in the Neonatal Pituitary T h e a b o v e m e n t i o n e d decrease in pituitary o u t p u t could be due to an inability o f the n e o n a t a l pituitary to produce adultlike quantities o f A C T H . I m m u n o c y t o c h e m i c a l d a t a suggest that all P O M C - p r o d u c i n g cells in the a n t e r i o r pituitary o f the day-old p u p are capable o f cleaving the precursor molecule (72,181). However, proteolytic processing is far less extensive at this age t h a n in the adult ( 4 0 - 5 0 % o f the precursor is not cleaved); even by weaning age there is a substantial a m o u n t o f precursor-sized material. T h e r e are additional differences in n e o n a t a l P O M C processing (e.g., substantial a m o u n t s o f c~MSH-sized material, only present in m i n i m a l a m o u n t s in the

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FIG. 3. Plasma corticosterone levels ~g/dl) in 4-day-old rat pups 2 h and 4 h following injection of vehicle or different doses of ACTHt_24. Pups were either nondeprived (NDEP) or maternally-deprived (DEP) for 24 h. During deprivation period pups were kept at 30-33°C. At the end of the deprivation period (or at the corresponding timepoint for NDEP pups), pups were weighed and injected ip with vehicle (0.90'/0 NaCI, in a volume of 0.1 ml/10 gbw) or ACTHI.24 at one of four different dilutions (0.01, 0.10, 1.0, or 10 IU/kg bw, in a volume of I% bw). Two or 4 h later the pups were rapidly removed from their home cage and trunk blood was collected in heparinized 1.5 ml microcentrifuge vials. Blood was centrifuged at 2000 rpm for 20 min, following which plasma was extracted and frozen at - 2 0 ° C until subsequent radioimmunoassay of CORT (ICN Biomedicals). Data were analyzed by means of a 2 (Conditions: NDEP, DEP) x 5 (Doses: 0.00, 0.01,0.1, 1.0, I0.0) × 2 (Timepoints: 2h, 4h) ANOVA, since sex was not a significant variable. CORT values in NDEP animals did not differ between timepoints (i.e., 2 h = 4 h). This was not the case for DEP animals: All ACTH-injected pups had higher CORT levels at 2 h than at 4 h. Values correspond to the mean ± SEM. •17 < 0.05, compared to NDEP pups; "I'P < 0.05, compared to vehicle-injected pups.

558 however, result in a concomitant difference in basal levels of plasma ACTH, which do not change substantially throughout ontogeny (156,178,180,190). More importantly, the pituitary has been shown to secrete massive amounts of ACTH under certain specific conditions. Plasma ACTH levels of around 2500 pg/ml, for example, have been measured 30 min following high doses of norepinephrine 8-day-old pups (55), and similar values have been achieved following surgical or chemical ADX (54,177,179). Synthetic capacity therefore does not appear to be a limiting factor during ontogeny.

Inhibition of A CTH Secretion ACTH secretion at any given moment reflects the balance of inhibitory and stimulatory signals acting upon the corticotroph. It follows, then, that a high degree of inhibition would result in a decrease in pituitary output. An enhanced negative feedback system at the pituitary level has, in fact, been postulated to underlie the neonatal decrease in stress-induced ACTH output (128,130,179). However, as we shall see in the following sections, the data do not necessarily support this hypothesis. Pituitary response following adrenalectomy. The increase in ACTH secretion following removal of CORT (either by surgical or chemical adrenalectomy) is commonly used as a measure of the feedback operating in the intact animal. A number of studies have examined the effects of short-term and long-term ADX on basal and stress- or CRF-induced ACTH secretion at different ages throughout ontogeny (54,177, 179,186). Although the picture is by no means complete, some generalizations appear to be emerging, at least with regards to changes in basal levels following ADX: (a) Plasma ACTH levels following surgery follow a biphasic pattern. Thus, ACTH concentration increases rapidly after the operation, peaks ( - 1 5 0 0 pg/ml) at around 3 hours, and then subsequently decreases over the next 12-24 hours. Forty-eight hours after ADX, however, levels are once again as high as at the initial peak, and they seem to remain stable thereafter (177). (b) This pattern does not appear to be age-dependent, at least with regards to peak values (54,177,179,186). (c) Both the pattern and the magnitudes of the ACTH response to ADX closely resemble those found in adults (31,32,64). The available data on the effects of ADX on stress- or CRF-induced ACTH secretion are less consistent (179,186). The results of Walker et al. (179) and Widmaier (186) indicate that both stimuli result in a further increase in ACTH levels in ADX pups. However, the magnitude of this increase depends on the nature of the challenge. In addition, whether it is age-specific a n d / o r dependent on the length of time elapsed between ADX and testing remains unclear, since (a) neither study included age as a factor, and (b) Walker et al. (179) used 24 h-ADX pups, whereas Widmaier (186) tested pups 72-h following surgery; as mentioned above, these represent very different stages of the infant's response to ADX. As mentioned previously, an enhanced negative feedback has been proposed to underlie the SHRP (128,130,140,179). The marked increase in ACTH secretion observed following ADX in the neonate has been interpreted as supporting the notion of enhanced pituitary feedback (179). Although this may in part be true, as we shall point out, there remain a number of problems with this interpretation. The rapid increase in ACTH secretion that occurs following surgical or chemical ADX appears to be too fast to be genomically-mediated (64). In the adult animal, ADX leads to a rapid release of CRF and AVP from hypothaiamic terminals in the median eminence into portal circulation; this is closely

ROSENFELD, SUCHECKI AND LEVINE followed by a large increase in plasma ACTH. This initial hypothalamic secretion is probably controlled by other neural inputs acting upon the CRF and AVP terminals (GABA, norepinephrine, opioids); GCs have a non-genomic inhibitory action upon these inputs (66,69,151,187, for reviews see 45,67). Increase in synthesis of the respective mRNAs (resulting from decreased GR-mediated feedback) only occurs at a later time point (163). A similar process is very likely occurring in the infant. The decrease in ACTH secretion which follows the initial peak (i.e., between 3 and 48 h, approximately) has been generally ascribed to pituitary depletion of stores (31,32,177). However, this is hard to reconcile with the fact that a 24-h ADX pup, presumably with a depleted pituitary, can increase ACTH secretion 4-fold within 30 minutes of CRF administration (179). Once again, these data suggest that ACTH secretion during the first 24 h following ADX involves more than a simple release from GR-mediated pituitary feedback. As from 48 h onwards, the ADX-induced increase in ACTH secretion probably does represent the absence of genomically-mediated feedback, at least in the adult (64). However, although basal (i.e., non-stimulated) levels of ACTH are extremely elevated in 48 h-ADX pups, they are no more elevated than in similarly-treated adults (31,32,54,64,177,186). Thus, whereas these data certainly support the existence of negative feedback in the pup, they do not in any way indicate that this feedback is enhanced. Grino, Burgunder, Eskay, and Eiden (49) reported that 48 h-post-ADX, pnd 7 pups show a smaller increase in POMC mRNA than their pnd 14 counterparts (3- and 7-fold, respectively), suggesting that, if anything, the reverse is true. The increased magnitude of the stress- or CRF-induced ACTH response found in ADX pups, again, should be interpreted with caution. First, only one study has examined this issue in longterm ADX pups (186). This study found a relatively small difference between ADX and intact animals with regards to the absolute magnitude of the change induced by 2-DG (an increase of - 1 3 0 and - 4 0 pg/ml, respectively). Walker and coworkers (179) found much larger differences in absolute magnitude following ether stress or vehicle or CRF injection. However, testing took place 24 h after ADX; as discussed above, other processes in addition to classical GRmediated feedback are probably occurring at this time. Finally, in the adult animal, CORT acts both on the pituitary and the brain (hypothalamic and suprahypothalamic sites) to turn off its own secretion [for reviews see (30,35, 71,113)]. However, adult rats with anterolateral hypothalamic deafferentiation show no increase in POMC mRNA following ADX (33). This suggests that in the adult at least, brain (and not pituitary) disinhibition is primarily responsible for the ADX-induced increase in ACTH (33,86). The studies reviewed in this section indicate merely that ADX leads to an increase in ACTH secretion; this could be due to a decrease in the inhibitory signal at the pituitary level. An equally plausible explanation, however, is that feedback was eliminated at the brain level; this would lead to increased CRF a n d / o r AVP release. These, in turn, would induce ACTH hypersecretion. In summary, data obtained from ADX studies indicate that GCs exert a chronic inhibitory effect on ACTH secretion in the neonatal rat. They do not, however, allow a discrimination to be made as to the precise site or mechanism (i.e., pituitary or brain, genomic or non-genomic) at which this feedback takes place. More significantly, they provide no evidence to support the notion that this system is in any way enhanced during this period. Rather, the inhibitory effect is very similar in magnitude to that found in adults. Since adults are perfectly

HPA AXIS AND DEVELOPMENT capable of responding to stress under these conditions, it is unlikely that negative feedback, on its own, can account for the hyporesponsiveness characteristic of the SHRP. Pituitary response following steroid treatment. If GCs are administered to intact adult animals some time prior to pituitary stimulation (e.g., stress, CRF), the ACTH response to the stimulus is markedly reduced. This decrease is assumed to be due to the GR-mediated inhibitory effect of GCs at brain and pituitary feedback sites. The extent of inhibition is a function both of the dose (and type) of GCs used, and the number of feedback sites available [(14,152) for review see (71)]. A similar paradigm has been used to examine the negative feedback potency of CORT during ontogeny (179). Thus, pups were pretreated with either 1 mg/kg CORT or dexamethasone [(DEX) a synthetic GC that does not bind to CBG]; plasma ACTH levels were measured 30 min following exposure to urethane. Whereas DEX completely eliminated the response at all ages tested (from pnd 5 to 21), CORT was only partially effective at pnd 10, and totally ineffective at later ages. A subsequent dose-response study indicated that pnd 18 animals require a 5-fold larger dose of CORT than pnd 10 pups in order to suppress the ACTH response. In contrast, there was no age-related difference in the dose of DEX required. In vitro, the ICs0 for CORT inhibition of CRF-induced ACTH release from whole pituitaries was greater at pnd 2223 than at pnd 3-5. As in vivo, the ICs0 for DEX did not show a significant change with age (179). The decrease of CORT potency with age has been postulated to reflect concomitant increases in CBG (179). While the lack of any age differences with DEX suggest this may in part be the case, there are a number of problems with this interpretation. First, CBG concentrations are similar at pnd 5 and 10 (170,177), then begin to rise slowly, but at pnd 14 are still only about one-third of those found in the 3-week-old pup (57). In contrast, CORT has a greater suppressive effect on urethane-induced ACTH secretion in 5-day-old pups than in 10-day-old pups; these in turn show a larger suppression than their 14-day-old counterparts. There is no difference between this latter age and older (i.e., pnd 18 and 21) pups (177). This pattern differs substantially from that shown by CBG, suggesting that changes in CBG are not solely responsible for the changes in CORT potency. Thus, whereas variations in CBG levels may be partially responsible for the observed changes, it is clear that other factor(s) are also involved. Furthermore, as stated in the previous section, at least in the in vivo studies, the reduced ACTH secretion may be an indirect consequence of increased GC inhibition on the brain. In a similar vein, Walker and colleagues (177) demonstrated that subcutaneous CORT pellets implanted at the time of ADX are sufficient to prevent the ADX-induced increase in ACTH (see above). In this case, however, no comparison was made across ages. In addition, plasma CORT levels in pellet-implanted pups were not constant throughout the study: there was a 100-fold drop (i.e., from - 2 5 to - 0 . 2 5 #g/dl) over the 5-day period of the experiment. Thus, it is hard to arrive at any conclusion other than the previously stated one that CORT feedback is operational in the infant rat. The above studies indicate that CORT exerts a chronic inhibitory effect on its own secretion throughout development. They do not prove that this feedback is enhanced in a physiologically relevant manner, nor that it can account for neonatal hyporesponsivity.

Pituitary Response to Hypothalamic Secretagogues In the adult animal, a variety of hormones, including CRF, AVP, angiotensin II, and norepinephrine act upon the cortico-

559 trophs to induce ACTH secretion. Of these, CRF and AVP appear to be the primary determinants of release [for reviews see (5,30,107)]. On its own, AVP has only a relatively weak effect; however, it synergizes potently with CRF (10,47, 166,169). The diminished ACTH response to stress found during development might be postulated to be due to a decreased pituitary responsiveness to hypothalamic releasing factors, and/or a decreased presence of these factors. The former hypothesis can be tested by directly administering these secretagogues, and examining the effects on ACTH secretion. In vivo studies have yielded conflicting data. On the one hand, Guillet and Michaelson (50) reported no age-specific differences following treatment of 1-, 7-, 14-, and 21-day-old rat pups with a median eminence extract. Walker and colleagues (178), in contrast, found a 5-fold increase in ACTH secretion in response to synthetic oCRF over this same age span. These discrepancies might be due in part to the different test stimuli used (median eminence extract versus ovineCRF), and/or to the different sampling times used (5 min versus 30 min). In vitro studies, however, do not support an age-dependent increase in pituitary responsiveness to CRF. First, isolated pituitaries taken from 7- and 14-day-old pups do not differ with regards to the amount of/3-endorphin (which is co-released with ACTH) released following incubation with CRF (49). Second, pituitary concentrations of CRF receptors show a transient overshoot in the first week of life (2.4-fold adult values at pnd 5), but by pnd 10 no longer differ from those of adults. Furthermore, the affinity of the receptor for its ligand does not seem to change with age (178). Thus, if there is in fact an age-dependent increase in pituitary responsiveness to CRF, it is most likely due to post-receptor mechanisms. Whereas the response to CRF does not appear to be fundamentally impaired during development, in vitro experiments suggest the opposite may be the case for AVP. In cultures of anterior pituitary cells of 10-day-old pups (in contrast to cultures of adult cells), AVP does not trigger sustained ACTH secretion. AVP is also less potent in synergizing with the effects of CRF on both ACTH secretion and cAMP formation. These differences are due to a low density of receptor sites combined with a reduced effectiveness of the post-receptor signal transduction process (76). The available evidence therefore suggests that, during development, stimulatory inputs to the pituitary (particularly AVP) may not have adult-like effectiveness in causing ACTH release. The possibility that during this period there is in addition a decrease in these stimulatory inputs shall be examined in subsequent sections. In summary, it seems reasonable at this point to propose that during development: (a) the potential secretory capacity of the pituitary is not reduced, insofar as certain experimental paradigms (e.g., ADX, bacterial endotoxin) are capable of inducing massive ACTH secretion, (b) removal of GCs leads to a large increase in pituitary output; however this increase is no greater than that found in the adult organism following a similar procedure, and (c) the pituitary is responsive to CRF, although it may be refractory to AVP. Taken as a whole, thus, the data do not support a major role for the pituitary in determining neonatal stress hyporesponsiveness. BRAIN LEVEL The presence or absence of regulatory mechanisms at the lower level of the HPA axis would be of little consequence if hypothalamic cells did not secrete their appropriate releasing factors. This will depend on the capacity of the hypothalamic cells to synthesize, transport, and secrete these peptides. As in

560 the pituitary, it will also be a function of the relative strength of the inhibitory and stimulatory signals acting upon these cells. In the adult animal, a wide variety of compounds act as "releasing factors" (e.g., AVP, angiotensin II, cholecystokinin, vasoactive intestinal polypeptide) [for reviews see (5, 137,161)]. Of these, CRF appears to be the most potent, although AVP, a weak secretagogue on its own, strongly potentiates the action of CRF [47,115,166,169,191, for review see (114)]. The cells that constitute the principal source of CRF to the hypophyseal portal vasculature are located in the dorsal medial part of the parvocellular division of the PVN [(158,162) for reviews see (136,159,161)]. Their axons project to the external lamina of the median eminence (81,189). Many, if not all, of these cells are capable of expressing AVP, as well as one or more of the abovementioned biologically active peptides [(59,74,96,139,165) for reviews see (137,160)]. AVP is also present in magnocellular neurons in the PVN (171) and supraoptic nucleus of the hypothalamus (SON) [for review see (161)]. These magnoceUular sources give rise to projections that course through the internal lamina of the median eminence on their way to the posterior pituitary, where they release their products into systemic circulation (11,15,143,155,176). However, recent evidence suggests this magnocellular secretion may also gain access to the portal vasculature (via exocytotic release at the level of the median eminence) (20,60), a n d / o r may affect the corticotropes directly (via vascular links between the posterior and anterior pituitary lobes) (105). There is a noticeable paucity of information regarding these issues during ontogeny, largely due to the technical difficulties inherent in measuring a n d / o r interpreting changes in brain neuropeptides. Prior to discussing the actual data, we would like to point out some of the more salient problems which should be taken into account when interpreting the data. First, stress-induced neuropeptide secretion takes place in the order of milliseconds, rather than minutes as is the case for ACTH and CORT (175). Small differences in the timing of testing and sampling may thus constitute a large source of experimental variability. Second, there are age-dependent changes in volume of distribution of the neuropeptide, correlating with the maturation of the portal vasculature (184). Thus, even a small absolute quantity of neuropeptide might result in a portal concentration in the pup comparable to that found in adults. However, given current technology it is virtually impossible to measure hormone levels in neonatal portal circulation. Third, the means with which to evaluate the dynamics of neuropeptide secretion into the portal vasculature of the neonate are not yet available. Measures of tissue content, which at this point constitute the only available alternative, are hard to interpret. A decrease in tissue content, for example, could reflect lowered peptide synthesis. However, it could also indicate greater release, or a combination of both processes. Conversely, increased content could indicate greater synthesis and/or reduced release. Furthermore, low (as compared to adult) basal levels do not necessarily imply the system is incapable of secreting large amounts of hormone [note that pituitary ACTH content in the neonate is a fraction of that found in the adult organism, yet the pup is capable of massive ACTH output under specific circumstances (190)]. Fourth, in the adult hypothalamus there is a significant proportion of CRF-containing cells that do not release their content in the median eminence; rather, they give rise to a widespread neural network in which CRF may act as a neuro-

ROSENFELD, SUCHECKI AND LEVINE transmitter or neuromodulator [(2,46,94,103, for review see (15)]. An unknown fraction of this non-hypophysiotropic CRF may be included in measurements of content in whole hypothalamus. Analogous problems may arise when attempting to assess AVP levels in the neonate. Fifth, examination of mRNA levels allows for more precise anatomical resolution and may provide insight on the regulation of peptide synthesis. However, mRNA accumulation and peptide content appear to be dissociated, at least in the case of hypothalamic CRF and AVP in the neonate (43). Finally, the data on hypothalamic and extrahypothalamic binding sites for GC receptors in the neonate remain far from clear (80,92,93,102,118,119,120,131,167). Little is known, for example, about non-genomic mechanisms of CORT action, through which some of the negative feedback effects of CORT may be mediated (104,164). Taking the above caveats into consideration, in the following sections we shall attempt to summarize what is known on the postnatal ontogeny of (a) hypothalamic CRF and AVP content under basal conditions and following stress, and (b) some of the factors involved in regulating H P A activation at the brain level.

Basal Levels of Hypothalamic Secretagogues Although there is some variability with regards to the absolute magnitudes of peptide measured, overall the data on in vivo basal content of hypothalamic CRF provide a fairly consistent picture. Thus, CRF content is very low ( - 1 0 0 p g / hypothalamus) soon after birth, and increases steadily thereafter (18,19,29,43,61,123,179). However, levels at the time of weaning ( - 7 0 0 pg/hypothalamus) are still 40-5007o below those found in adults (123,179). It should be emphasized that these values correspond to total hypothalamic content, and are not concentrations. Basal levels of CRF mRNA in the PVN follow a similar ontogenetic pattern (48). "Basal" CRF release has also been examined in an in vitro model in which isolated complete hypothalami are incubated in a defined medium (Krebs buffer). Somewhat surprisingly, CRF secretion under these conditions does not seem to rise with age, at least between pnd 9 and 24 (184,186). It is not clear whether these levels are equal to those found in adults (184), or smaller (186). The differences with the in vivo data may simply represent an in vitro artifact. Alternatively, however, they may indicate that extrahypothalamic structures are important in the regulation of CRF synthesis/output in vivo. Basal levels of AVP in hypothalamus and median eminence remain extremely low (i.e., < 1007o of adult) until pnd 14, approximately (43,123,150). At this point they begin to increase, but at the time of weaning they are still less than 2007o of adult values (123,150). The main increases in peptide thus seem to take place late in development. Levels of AVP mRNA in hypothalamus/median eminence show an earlier and more gradual increase with age: They double between the end of the first and second weeks of life (from 20 to 40070 of adult), and by pnd 21 are about 55070 of adult control. These increments take place following cessation of neuronal cell proliferation; hence, they are probably due to an increase in gene expression and not in cell number (4). It is logical to expect mRNA levels to increase prior to those of their respective protein. However, in this case there is a very marked lag between both processes. Thus, although AVP concentrations per se are extremely low throughout the SHRP, the pup does have during this period a fairly substantial pool of mRNA. This may result from immaturity or inhi-

H P A AXIS AND DEVELOPMENT bition at the translational level, as has been demonstrated for oxytocin (3). Finally, the differential ontogenetic patterns of CRF and AVP are reflected in the ratio of hypothalamic/median eminence AVP to CRF. This ratio, which is about 10:1 in the first few days of life, decreases to as low as 3 : 1 during the SHRP, and subsequently increases slowly. By pnd 21, however, it is still only 10 : 1, substantially below the adult ratio (40 : 1) (123). As mentioned previously, AVP has potent synergistic effects on CRF-induced A C T H release. The possibility has been raised that a certain ratio of released AVP to CRF is required for "normal" ACTH release (123). The diminished ACTH response to stress during the SHRP may therefore be a consequence of these low ratios, rather than of diminished CRF secretion per se (123). In this regard, it is interesting to note that in the adult animal there is increasing evidence that the main role of CRF is to set the "stimulatory tone" on the corticotropes. The situational "fine-tuning," in contrast, would be determined by the relative abundance and type of co-secretagogues (e.g., AVP). (34,108).

Stress-Induced Levels of Hypothalamic Secretagogues Little information is available on this subject. Walker, Scribner, Cascio, and Dallman (180) measured changes in hypothalamic CRF and AVP content in pnd 10 pups during 12 h of maternal deprivation (at room temperature), or 1 h cold stress (at 4 ° C) followed by 11 h of maternal deprivation. Their results appear to indicate that both CRF and AVP content increase rapidly (i.e., within 5 min) following separation, and remain elevated for at least 12 h. Additional cold stress did not further affect CRF levels, but decreased AVP levels towards basal values. Increased content is generally used as an indication of decreased secretion. However, it is hard to understand how the increased ACTH and CORT secretion that follow maternal deprivation (see previous sections), could result from decreased CRF and AVP secretion. Furthermore, a stressor should activate, not inhibit, the H P A axis. Alternatively, the elevated contents may indicate an augmented synthesis combined with a deficient transport a n d / o r release mechanisms. Changes in synthesis, however, cannot be measured within such a short time frame. The relative decrease in AVP content in deprived/cold (as compared to deprived) animals could be interpreted as increased cold-induced AVP secretion. However, this decrease was more noticeable in the period following the cold stress, and not during the cold stress itself. The meaning of these data thus remains obscure. Using a different approach, Widmaler examined the dynamics of CRF secretion in response to a specific stressor (glucoprivation) in an in vitro model [see previous section, (184,186)]. Isolated hypothalami taken from adult animals show a marked increase in CRF release when incubated in severely hypoglycemic (0.55 mM glucose) or glucopenic (22 mM 2-DG) conditions. In contrast, no such increase is seen in hypothalami taken from rats 24-days of age or younger (184,186). In fact, even a depolarizing concentration of KCI does not stimulate CRF release at the younger ages (184). This would seem to indicate that the neonatal rat hypothalamus has minimal stores of releasable CRF.

Regulation of CRF and A VP Secretion In the adult animal, the PVN cells appear to constitute the final central integrators of the organism's response to stress [(17,63,83) for reviews see (135,160)]. As such, they receive

561 neuronal and hormonal inputs from multiple central and peripheral sources. Neural inputs to the PVN. Neural inputs to the hypophysiotropic cells of the PVN can be divided, according to their modality, into five general categories: visceral, somatic/special sensory, circumventricular, intrahypothalamic, and limbic (135). Many of the principal afferents express multiple neuroactive agents [(85,138) for review see (135)], and synapse with specific subpopulations of parvocellular cells in the PVN. Different stressors may therefore activate distinct subsets of neurons, which may in turn result in a particular pattern of secretagogue release. It should be emphasized that these inputs do not project exclusively to the hypophysiotropic cells; on the contrary, most of them have a wide variety of targets (including the magnocellular cells and other hypothalamic nuclei) [(136) for review see (135)]. This anatomical/neurochemical organization allows for a complex integration and coordination of the autonomic, endocrine and behavioral responses to stress. The ontogeny of afferent control of the PVN remains largely unexplored. However, there appears to be some evidence (albeit circumstantial) that differential development of these pathways may at least partly explain the idiosyncracies of the SHRP. In the adult, visceral information (which includes glossopharingeal and vagal sensory information) projects to the nucleus of the solitary tract (NTS), from whence it is relayed to the parvocellular CRF and magnocellular AVP neurons via highly differentiated ascending catecholaminergic fibers (28, 129). Mesencephalic and pontine projections to the CRF neurons (the latter projection, probably cholinergic) have been proposed to carry somatic/special sensory information (84,124). The effects of circulating angiotensin II are transduced by the subfornical organ; this circumventricular structure has a direct projection to both the parvocellular and magnocelhilar areas (135). Many hypothalamic cell groups innervate the PVN; the functional implications of these connections remain for the most part obscure. Finally, limbic structures, which mediate neocortical influences (i.e., "higher" integration of information from the external and internal environment) on the hypothalamus, differ from the above in that they do not have any major direct projections to the PVN. They are, however, connected to the bed nucleus of the stria terminalis, which projects strongly to the PVN (101,136,157). The rat is an altricial species in which much of the central nervous system neuronal and glial growth and differentiation takes place postnatally (58). The extent and rate at which maturation occurs varies widely in different brain areas (58). It is not unlikely, then, that the various pathways that mediate afferent inputs to the hypophysiotropic cells become functional at different times during ontogeny. Although this has never been examined systematically, there are some indications that this might well be so. The biochemical and morphological development of the monoamine (MA)-contalning neuronal system, for example, is largely postnatal. Adult brain concentrations of MA are only achieved around pnd 20-30, depending on the area (70,90). There is a concomitant postnatal morphological maturation and proliferation of the axon terminal apparatus, the rate of which is also site-specific (90). Catecholaminergic synapses on hypothalamic magnocellular neurons are established mostly during the first few weeks of life; however, the maturation of the PVN lags behind that of the supraoptic nucleus (73). Finally, adult concentrations of both cd and c~2 adrenergic receptors are not achieved until approximately pnd 28 (99). As stated above, both CRF and AVP release in response to

562 certain stimuli (e.g., baroreceptor and other visceral stimuli) is mediated by central aminergic pathways. In the 8-day-old infant, NE injection enhances the pituitary response to ether stress (55). This would suggest that the diminished response to this stressor seen in control animals is at least partly due to immature NE pathways. The absence of an in vitro CRF response to glucoprivation during the first month of life [see Stress-Induced Levels of Hypothalamic Secretagogues (184)] is also suggestive of immature (or inhibited) afferent inputs to the PVN. In the adult, there are both intra- and extra-hypothalamic glucose sensors. The former is (are) probably located within the ventromedial nucleus (VMN) of the hypothalamus, and may activate CRF secretion via one or more serotonergic a n d / o r cholinergic interneurons (188). The latter probably involve the NTS, which has a direct catecholaminergic projection to the PVN [for review see (109)]. One possible explanation for the in vitro lack of response in the neonate is that the intrahypothalamic glucose sensors a n d / o r the pathways connecting them to the hypophysiotropic cells are not yet developed. Both serotonergic and cholinergic neurons show extensive postnatal maturation (27,82,122); either of these neurotransmitters might therefore be the rate-limiting factor. Alternatively, glucose sensors may be exclusively extrahypothalamic during development. This could account for the lack of response in vitro, since these inputs would obviously be absent in the in vitro preparation. The relatively small response to glucoprivation seen in vivo might be induced by other hypothalamic factors (e.g., AVP), and/or might reflect immaturity of the neural pathways connecting the extrahypothalamic glucostat with the PVN (e.g., the inputs to the PVN from the NTS are catecholaminergic; as mentioned previously, these pathways show a delayed development). Finally, those stressors that do elicit a relatively large pituitary response in the neonate, either act directly on the PVN (e.g., interleukin), a n d / o r do not require collative processing (e.g., ether, urethane) (9,17,21,53,132,153). The response to stimuli that do require this kind of processing (e.g., novelty) is presumably mediated by limbic structures, in particular by the hippocampus [for review see (148)]. Hippocampal neuroanatomical and functional maturation occurs late in ontogeny (i.e., largely between pnd 15 and pnd 28) (8,79, 97,144). Although this clearly does not constitute proof that the "deficit" seen in the neonate originates at the brain level, it does provide strong circumstantial evidence that this may be the case. Hormonal inputs to the PVN. In the adult animal, glucocorticoids exert a potent negative feedback effect on both the synthesis and release of hypothalamic secretagogues [for reviews see (35,52,113)]. This effect is a consequence of direct actions of the GCs on the hypophysiotropic cells themselves, and indirect actions on other brain areas, particularly the limbic system [(22,75,77,112,168,174) for reviews see (100,161)]. To what extent this is also true in the neonate remains a matter of debate. In the adult, ADX results in a rapid decrease in hypothalamic CRF content, followed about 48 h later by a massive increase (31,163,182). The initial decrease probably reflects release of CRF from the terminals, whereas the subsequent increase is paralleled by augmented CRF mRNA levels and, therefore, probably represents augmented synthesis (110,163). Twenty-four h after ADX, the 5-day-old infant also shows reduced hypothalamic levels of CRF (179). However, 48 h after the operation there is no change in mRNA levels in 7-

ROSENFELD, SUCHECKI AND LEVINE day-old pups, although there is a 2-fold increase in pnd 14 animals (49). The above data would seem to indicate that GR-mediated CORT feedback on the CRF cell (which results in an inhibition of CRF mRNA synthesis) is much reduced or absent during early ontogeny. Additional support for this conclusion comes from in vitro studies showing that isolated hypothalami obtained from pnd 8-10 pups ADX 72 h prior to sacrifice do not release more CRF (either under basal or glucopenic conditions) than those obtained from intact or sham-operated animals 086). Finally, GR concentrations in the hypothalamus are extremely low during this period, again suggesting that the brain is not a major target of direct GR-mediated GC feedback until later in ontogeny (102). ADX does, however, lead to an initial increase in CRF secretion (as reflected by the post-surgical decrease in hypothalamic content). In the adult, there is evidence that GCs can act at the level of the CRF terminals to inhibit peptide release (1,68,69,126). Although the abovementioned data suggest a similar process may be taking place in the infant, this hypothesis remains to be tested. In summary, the available data suggest the central components of the H P A system show extensive postnatal maturation. Thus, during early infancy the hypophysiotropic cells themselves may show diminished capacity to synthesize, store and secrete neuropeptides. Furthermore, the neuroendocrine network which mediates and integrates the stress response in the adult, appears to be only partially functional even by weaning age. Central H P A responsiveness may, in addition, be under other forms of chronic inhibition (e.g., by maternal factors). DISCUSSION

The SHRP was originally defined on the basis of the absence (or pronounced decrease) of a CORT response to either stress or direct stimulation of the adrenal with ACTH (145). A closer examination indicated that this diminished output is not limited to CORT secretion; rather, it extends to all levels of the H P A axis. Underlying this, is a multifactorial regulatory system, which in turn depends on both internal and external inputs (e.g., neural and maternal signals) to maintain overall quiescence. The low basal and stress-induced concentrations observed at different levels of the axis appear to be the result of different but concomitant processes: 1. H y p o t h a l a m u s - t h e low basal and stress-induced levels of CRF and AVP may be due to immaturity (especially of those neural pathways that provide stimulatory inputs to the hypophysiotropic cells), and perhaps also to chronic maternal inhibition. The mechanism(s) underlying this inhibition have not yet been examined. The ontogenetic changes in the stimulus-specificity of the stress response may reflect concurrent maturation in the specific pathways that mediate the effects of each particular stimulus. 2. Anterior pituitary--both basal and stress-induced levels of ACTH are probably mostly a reflection of hypothalamic stimulation. There also appears to be a chronic inhibition by GCs; however, this inhibition does not seem to be any greater than that found in the adult animal. 3. P e r i p h e r y - b a s a l concentrations of plasma CORT appear to reflect variations in metabolism/volume of distribution of the steroid, due to changes in CBG levels (but not immaturity of steroid-synthesizing machinery). In addition, basal as well as stress-induced levels of adrenal and plasma

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CORT reflect ontogenetic changes in adrenal sensitivity and steroidogenic capacity that are determined both by maternal factors (perhaps indirectly via a "priming" effect of augmented ACTH levels) and other endogenous factors (as yet uncharacterized). The above data suggest that the main rate-limiting sites for hormone secretion during development are at the brain and adrenal levels. The brain exerts a selective regulation, in that it only 'permits' certain specific signals (i.e., stressors) to be processed. The adrenal response, in contrast, does not appear to be stimulus-specific. During the SHRP, even very high levels of ACTH fail to elicit more than a minimal CORT response (117,178,180,186,190). Both basal and stress-induced CORT secretion show a Ushaped ontogenetic curve; this, in fact, is what gave rise to the definition of SHRP. ACTH, CRF, and AVP secretion, in contrast, either remains the same or increases gradually over this same period. This suggests that the proximal cause of the SHRP (as originally defined) rests at the adrenal level, and is primarily a consequence of the lack of responsiveness of this gland to ACTH. This unresponsiveness may, of course, be due to centrally-mediated phenomena (e.g., ACTH priming). During the SHRP, the HPA system is potentially capable of releasing massive amounts of ACTH (and, presumably, hypothalamic secretagogues), but not CORT. The argument has been made that in the absence of CBG these low levels of total CORT would have a very high biological potency, since most of the hormone would be in its free (biologically active) form (130,179). However, the concentration of free CORT in the infant is at least 10-fold lower than that of the adult (57). In consequence, during the SHRP concentrations of CORT (both in its bound and free forms), but not necessarily ACTH or CRF/AVP, are maintained at low and remarkably constant levels. The teleological implications of this dichotomy remain speculative. Low levels of GCs are required for the normal differentiation of various peripheral tissues [for reviews see (7,95)]. In addition, they modulate cellular differentiation and neurotransmitter expression in various areas of the brain, the autonomic nervous system and the adrenal medulla [for reviews see (12,38)]. High levels, in contrast, have overall catabolic effects: They inhibit cell division, protein synthesis, and amino acid and glucose uptake [for reviews see (95,100)]. Furthermore, they have been shown to alter specific developmental patterns in a variety of differentiating tissues, including the brain. A given developmental function is only sensitive to steroid action during a critical time period, after which it is refractory or responds in a reversible manner [for reviews see (7,12,38,95)]. The maintenance of low, fairly unperturbable levels of CORT during the SHRP supports the notion that

these organizational effects are critical for normal development. Recent research suggests that other hormones of the HPA axis may also have organizational effects dtiring development. Specifically, animals treated neonatally with high doses of CRF show less behavioral and physiological responses to stress as adults; this is not secondary to CRFinduced changes in GCs (62). An alternative function can be proposed for the "high ACTH/low CORT" state found in the neonate following certain treatments (e.g., endotoxin). As mentioned previously, the hyporesponsiveness of the neonatal adrenal may be at least partly due to a lack of ACTH trophic action. The stressinduced increase in ACTH in the neonate might then be hypothesized to be sufficient to "prime" the adrenal and, therefore, increase its responsiveness to subsequent stressors. Thus, the neonatal HPA system would be geared towards overall quiescence in terms of CORT secretion. However, it would have an option built in to begin responding if the disturbance (i.e., stress) were large enough, and persistent. CONCLUDING REMARKS In the preceding sections we showed that factors as diverse as synaptogenesis and maternal behavior may be important determinants of HPA activity in the neonate. Furthermore, we found that at every level of the axis, a number of regulatory mechanisms operate simultaneously. In some cases, these mechanisms appear to be closely interrelated. Thus, pituitary output may reflect, to a large extent, the concentration of hypothalamic secretagogues acting upon the corticotrophs. In other cases, such as adrenal sensitivity and secretory capacity, the regulatory mechanisms may be relatively independent. The ultimate goal appears to be to keep CORT levels within the narrow range of concentrations required for normal development. Both structural immaturity and active inhibitory processes appear to underlie the overall hyporesponsiveness of the system. Given the current state of knowledge, it would be naive to assume that any single factor is sufficient to account for the characteristic developmental pattern of the HPA system. ACKNOWLEDGEMENTS The research conducted in this lab was supported by grants HD02881 from National Institute of Child Health and Human Development, MH-45006 from the National Institute of Mental Health (NIMH), and U.S. Public Health Service Research Scientist Award MH-19936 from NIMH to Seymour Levine. Deborah Suchecki was supported by CNPq-Conselho Nacional de DesenvolvimentoCientifico e Tecnologico, grant 20.0154/90.7. The authors would like to thank Dr. E. R. de Kloet and Dr. T. Insel for their helpful comments on the manuscript, and Dr. J. Weinberg for assaying plasma CGB.

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