Ventromedial hypothalamic lesions inhibit corticosteroid feedback regulation of basal ACTH during the trough of the circadian rhythm.

We have determined the effects of bilateral electrolytic lesions of the ventromedial hypothalamus (VMH) on activity in the hypothalamo-pituitary-adrenal (HPA) system. Acutely, during the first 5 days, lesions of the anterior-medial VMH caused loss of the diurnal rhythms in food intake and plasma corticosterone (B) levels. Plasma B concentrations were elevated during the time of the normal trough of the basal diurnal rhythm in HPA axis activity and the diurnal rhythm in food intake was abolished, in agreement with the results of others. Consistent with hyperactivity in the HPA axis, lesioned rats had increased adrenal weight, decreased thymus and body weights and decreased plasma transcortin concentrations. To determine how lesions of the VMH provoke these increases in activity of the HPA system, the sensitivity of ACTH in adrenalectomized, lesioned rats to replacement with exogenous B was determined under basal conditions during the trough (morning-AM) and peak (evening-PM) of the diurnal rhythm in HPA axis activity. ACTH in lesioned rats in the AM was insensitive to feedback over the very low range of plasma B of 1-4 micrograms/dl, whereas sham-lesioned controls exhibited the normal, high sensitivity of ACTH to B at this time of day. There was no difference between the sensitivity of ACTH to this low range of B in the PM in VMH- and sham-lesioned rats. Two to 5 weeks after VMH lesions, as found by others, mean daily plasma B levels did not differ from sham-lesioned controls; however, plasma B during the AM was still mildly elevated in these rats. Inhibition of plasma B in the PM by dexamethasone was less effective in lesioned rats. Although HPA system responses to hypoglycemia, corticotropin-releasing factor and ACTH were normal, the lesioned rats exhibited obesity, hyperinsulinemia, hyperglycemia, hypertension and tachycardia, all signs consistent with mild hyperactivity of the PHA axis. Occupancy of type I, high-affinity corticosteroid receptors is known to control basal activity of the HPA system during the trough of the diurnal rhythm and to interact with glucocorticoid receptors to affect basal activity during the peak of the diurnal rhythm and during AM stress. We conclude that VMH lesions disrupt transmission of inhibitory signals, mediated by occupancy of type I corticosteroid receptors, that are initiated by a B feed-back site.

Chronic streptozotocin diabetes in rats facilitates the acute stress response without altering pituitary or adrenal responsiveness to secretagogues.

We have used streptozotocin (STZ)-induced diabetes in rats to determine whether this represents a sustained stimulus to the adrenocortical system and whether STZ-diabetic rats are able to mount an acute stress response. Furthermore, we compared pituitary responsiveness to CRF and/or arginine vasopressin, and adrenal responsiveness to ACTH in STZ- vs. vehicle-treated rats. We also compared the efficacy of dexamethasone inhibitory feedback in STZ-diabetic and control rats. Our results show that STZ-treated rats chronically hypersecrete corticosterone (B) as evidenced by their decreased thymus weights, their increased urinary B excretion, and their elevated mean plasma B levels during the light hours of the day. Despite the evidence for sustained hypersecretion of B, STZ-treated rats showed greater and more prolonged ACTH and B responses to the acute stress of histamine injection. However, when tested separately, neither pituitary nor adrenal responsiveness to their secretagogues were increased in STZ-diabetic compared to control rats. Dexamethasone inhibition of stress-induced B secretion was tested using two different paradigms: pentobarbital-anesthetized rats were given iv injections of acid saline, and awake rats were given ip injections of histamine. In both experiments the STZ-treated rats were relatively resistant to glucocorticoid inhibition of stress responses. This finding, taken together with the exaggerated ACTH and B responses to stress, strongly suggests that the facilitatory effects of chronic STZ-diabetes are a consequence of changes in sensitivity of central neural components of the adrenocortical system to stimulatory and/or inhibitory inputs, in conjunction with changes in glucocorticoid feedback sensitivity.

The pituitary-adrenocortical system of neonatal rats is responsive to stress throughout development in a time-dependent and stressor-specific fashion.

The responsiveness of the neonatal hypothalamus-pituitary-adrenal (HPA) axis to stress has been thought to be impaired or diminished during the first 2 weeks of life. Although we previously found full responsiveness of the hypothalamus-pituitary unit to adrenalectomy in young rats [days (d) 5-10], we failed to measure a significant increase in ACTH 10 min after ether administration until d14 of age. These studies were, therefore, designed to test the functional activation of the HPA axis after a single or repeated exposures to stress. Both qualitative (time-course, stressor-specific, circadian) and quantitative changes in the ACTH and corticosterone (B) responses to various stressors were tested during the first 10 days of life. Exposure to 3 min of ether vapor increased ACTH and B secretion (P less than 0.05-0.01) in 1-, 5-, and 10-d-old rats, with an increasing amplitude of both ACTH and B responses as a function of age. Peak secretion of ACTH occurred 5 min after the onset of stress (122 +/- 3.8 to 359 +/- 54 pg/ml on d1-10), while the time of maximal B increased as a function of age. Other stressors, such as maternal separation (12 h), cold (4 C; 60 min), or histamine injection (4 mg/kg BW, ip), provoked significant and stressor-specific ACTH and B responses in 10-d old rats. Histamine administration increased ACTH secretion above that of vehicle-injected rats, with a peak of secretion 15 min after drug injection (272 +/- 29 vs. 127 +/- 8 pg/ml; P less than 0.01). Histamine-induced B secretion peaked at 60 min (3.7 +/- 0.5 micrograms/dl). In contrast to early responses observed after ether, separation, or histamine stress, cold stress in 10-d-old pups caused a large ACTH and B release 4 h after the onset of cold compared to that in maternally deprived pups [ACTH: cold, 457 +/- 61 pg/ml; separated, 150 +/- 14 (P less than 0.01); B: cold, 3.3 +/- 0.4 micrograms/dl; separated, 1.8 +/- 0.2 (P less than 0.05)]. We did not detect morning-evening (AM-PM) differences in either the pattern or the magnitude of the ACTH or B response to maternal separation or cold stress. Suppression of cold-induced ACTH release by B injection (1 mg/kg BW) 2 h before stress was observed until 4 h after stress in the AM and PM, whereas when given after cold, B was less effective in the PM than in the AM at preventing the rise in ACTH levels observed at 4 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Stress-induced adrenocorticotropin secretion: diurnal responses and decreases during stress in the evening are not dependent on corticosterone.

To test whether the diurnal rhythm in stress responsiveness is dependent on corticosterone (B)-mediated negative feedback, the responses of intact (SHAM) and adrenalectomized (ADX) rats to restraint for 3-90 minutes or ip injection with saline in the morning (AM) and the evening (PM) were compared. In both SHAM and ADX rats, ACTH responses to restraint stress were larger in the AM. In intact rats, this could have resulted from both fast negative feedback, due to the rate of rise of B during the stress in the PM, and delayed negative feedback, due to the high basal concentrations of B before the stress in the PM. However, this diurnal pattern of stress responsiveness was not dependent on B, as the same relative responses to restraint and ip injection were found in ADX rats. To determine whether the lack of response of ADX rats in the PM to stress was due to a loss of sensitivity to endogenous secretagogues, ADX rats were given CRF + arginine vasopressin (AVP) while anesthetized with ether after 30 min of restraint. In both the AM and the PM, the pituitaries were able to respond to exogenous secretagogues. A second novel finding was that in the PM, but not the AM, plasma ACTH concentrations in the ADX rats decreased substantially during the period of restraint, despite the lack of B-mediated negative feedback. In the AM and the PM, ADX rats were restrained for 30 min and then stressed with ether for 6 min. The ACTH concentrations were not different before and after ether, suggesting that, although the pituitaries of ADX rats are able to respond to exogenous CRF + AVP after stress, an additional stress of ether exposure no longer stimulates endogenous CRF and AVP release after 30 min of restraint at either time of day. After 90 min of restraint in the AM and the PM, the relationship between ACTH and B was positive, not negative, providing no evidence of ongoing B-mediated negative feedback in the SHAM rats. Therefore, the same mechanism responsible for the decrease in ACTH secretion in ADX rats may occur in SHAM rats as well. From these results, we conclude that the diurnal rhythm in stress responsiveness and, in the PM in the ADX rats, the decrease in plasma ACTH during stress, are largely independent of B.

Regulation of basal ACTH secretion by corticosterone is mediated by both type I (MR) and type II (GR) receptors in rat brain.

The mechanisms involved in the physiology of the secretion of ACTH are reviewed. The secretion is regulated by the biological consequences of the occupancy of high affinity mineralocorticoid (MR) and lower affinity glucocorticoid receptors (GR) for corticosterone at specific sites of the rat brain. The regulation by this mechanism of basal secretion during the circadian rhythm, the effect of adrenalectomy and of corticosterone replacement is discussed. Experiments with RU486, a specific glucocorticoid antagonist, suggest that occupancy of both MR and GR is required for normal control of ACTH at the time of peak activity. The occupancy of the GR for a few hours per day apparently suffices to maintain steady levels of the products of GR-responsive genes throughout the body.

Adrenalectomy in the neonate: adult-like adrenocortical system responses to both removal and replacement of corticosterone.

Neonatal rats exhibit a period of diminished responsiveness to stress between days 3-10 of life, which has been shown to be associated with an increased sensitivity to corticosterone (B) inhibitory feedback. In this study we further investigated B feedback potency on regulation of ACTH by examining 1) the time course of changes in pituitary ACTH secretion and content, plasma B and B-binding globulin (CBG) concentrations, and thymus weight after adrenalectomy (ADX) performed on 5-day-old pups, with or without sc 5% B pellet replacement, and 2) the time required for acute (B injection) and the B dose required for constant (B pellet) inhibition of ACTH secretion in 10-day-old ADX neonates. As in adult rats, ADX in neonates caused an immediate (3 h) large increase (13-fold) in plasma ACTH levels compared to that in sham-operated rats, followed by a decrease by 12 and 24 h after surgery and a further and sustained increase during the next 4 days. Pituitary ACTH stores were diminished in ADX rats by 3, 12, and 24 h and increased thereafter. Five percent B pellet replacement abolished ADX-induced changes in plasma and pituitary ACTH until days 4-5, when plasma ACTH was slowly released from B inhibition (circulating B values were similar to ADX values). By day 10 of life, inhibition of plasma ACTH by calculated free B showed an IC50 of 1.09 nM. Plasma CBG concentrations exhibited a clear developmental pattern in sham-operated rats, being lower on days 6-8 than earlier or later. Typical ADX-induced increases in CBG levels were observed from day 3 on after surgery, at the same time as a transient decrease in CBG levels occurred in ADX plus 5% B rats. On day 10 of age, inhibition of CBG by calculated free B demonstrated an IC50 of 1.5 nM. Although no enlargement of the thymus was observed after neonatal ADX, thymus weight was significantly diminished by 12 h after B replacement and in a dose-related manner at 5 days with B pellets containing 5-25% B. The thymus contained mostly type II glucocorticoid receptors, which did not up-regulate 3 h or 5 days after ADX. Acute sc injection of B (10-34 micrograms/g BW) in 10-day-old rats inhibited ADX-induced ACTH secretion within 30 min, and the estimated half-time for the inhibition was 40 min. By 2 h after B injection, plasma ACTH levels were comparable to those in sham-operated animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacological evidence that the inhibition of diurnal adrenocorticotropin secretion by corticosteroids is mediated via type I corticosterone-preferring receptors.

These studies were performed to determine pharmacologically the corticosteroid receptor type that mediates the effects of corticosterone (B) on ACTH secretion in adrenalectomized rats. We have compared the effects of treating young male rats at the time of adrenalectomy and throughout the next 5 days with B, dexamethasone (DEX), or aldosterone (ALDO) in doses that elevated plasma levels to concentrations in the range between 0.2-30 nM. Plasma ACTH, corticosteroid-binding globulin (CBG), and thymus weight were measured in the morning or evening, and these steroid-sensitive end points were related to the circulating concentrations of B (total B - CBG-bound B), total DEX, and total ALDO. For the inhibition of ACTH the rank order of potency of the three steroids was B greater than DEX greater than or equal to ALDO in the morning (estimated IC50, 0.7 +/- 0.1, 2.3 +/- 0.5, and 4.9 +/- 1.6 nM for B, DEX, and ALDO, respectively). There was a significant shift to the right in steroid efficacy between morning and evening (estimated IC50 in the evening, 3.9 +/- 0.2 and 9.3 +/- 0.8 nM for B and DEX; ALDO at the concentrations achieved was ineffective). The rightward shift in efficacy may result from the circadian increase in drive to ACTH secretion. The rank order of potency for B and DEX on ACTH and the agreement between the steady state IC50 values achieved for these steroids and the Kd values determined for B and DEX with type I receptors in vitro strongly suggest that feedback control of basal diurnal ACTH by corticosteroids is mediated by association with type I, B-preferring receptors. By contrast, DEX was 3 times more potent than B on CBG (estimated IC50, 1.5 and 4.5 nM, respectively) and tended to be more effective on thymus weight, suggesting that the effects of corticosteroids on these peripheral targets are mediated by association of the steroids with type II glucocorticoid receptors. ALDO coinfused with DEX or B did not alter the inhibitory effects of these on ACTH, suggesting that ALDO does not interfere with these type I, B-preferring receptors in vivo. Because there is little if any evidence for type I corticosteroid receptors in the hypothalamus, these results strongly suggest that the majority of corticosteroid feedback inhibition of basal morning and evening ACTH secretion is mediated transynaptically by the activity of extra-hypothalamic neurons.

The adrenocortical system responds slowly to removal of corticosterone in the absence of concurrent stress.

After removal of corticosteroid feedback by surgical or pharmacological adrenalectomy, plasma ACTH increases more rapidly than can be explained by changes in receptor-mediated gene expression. In aminoglutethimide-treated rats, plasma ACTH increased only at doses much higher than those inhibiting plasma corticosterone, suggesting that adrenal enzyme blockers may themselves be stressful. To determine the adrenocortical system response to stressless corticosterone removal, adrenalectomized rats maintained for 5 days on corticosterone in the drinking water were switched to steroid-free fluid (-B) or again given steroid (+B); additional rats were adrenalectomized (ADX). Plasma ACTH did not differ between -B and +B rats until 18-24 h after steroid removal, regardless of whether steroid was withdrawn at the circadian maximum or minimum. Plasma ACTH was similar between -B and ADX rats 0.5-14 days after corticosterone removal, although morning plasma ACTH was more stable in -B rats at 4-7 days. Evening plasma ACTH increased significantly after day 3 in ADX and -B rats. Unlike ADX rats, -B rats did not exhibit pituitary ACTH depletion at 12 and 24 h, but both -B and ADX groups had significantly elevated pituitary ACTH by 6.5 days. We conclude that 1) rapid increases in ACTH secretion after surgical or pharmacological adrenalectomy result from interaction between stress and loss of corticosteroid feedback; 2) no immediate interaction occurs between loss of feedback and circadian stimuli; and 3) the effects of steroid withdrawal may require at least 3 days to be stably expressed.

Role of alpha-adrenergic mechanism in effects of morphine on the hypothalamo-pituitary-adrenocortical and cardiovascular systems in the rat.

The role of alpha-adrenergic mechanism in the acute effects of morphine in the hypothalamo-pituitary-adrenocortical (HPA) and cardiovascular (CV) systems, and the interrelationship between the HPA and CV responses to alpha-adrenoceptor antagonists and/or morphine were studied by peripheral administration of prazosin, a selective alpha 1-adrenoceptor antagonist, and yohimbine, a selective alpha 2-adrenoceptor antagonist, in conscious, unstressed or ether-stressed rats. The test substances were administered intravenously or intraperitoneally in chronically cannulated or noncannulated rats. In the i.v. experiment, morphine (1 mg/100 g BW) rapidly induced a pronounced bradycardia and a short-lasting fall in blood pressure (BP), followed by a rise in BP, and increased plasma corticosterone concentration. Prazosin (0.5 mg/kg BW) induced a rapid fall in BP and tachycardia, and increased plasma corticosterone concentration. Pretreatment with prazosin did not block the effect of morphine on the CV system, but abolished the morphine-induced increment in plasma corticosterone concentration. Yohimbine (0.5 mg/kg BW) induced a rapid and a subsequent slowly developing rise in BP and tachycardia, and increased plasma corticosterone concentration. Pretreatment with yohimbine did not block the effect of morphine on the CV system nor alter the stimulatory effect of morphine on the secretion of corticosterone. In the intraperitoneal experiment, morphine (2 mg/100 g BW) stimulated the secretion of adrenocorticotropic hormone (ACTH) and corticosterone and prazosin (1 mg/kg BW) stimulated the secretion of corticosterone, but pretreatment with prazosin reduced the morphine-induced increment in plasma corticosterone concentration in unstressed rats. In stressed rats, morphine reduced the stress-induced increment in plasma ACTH and corticosterone concentrations and prazosin also reduced the stress-induced increment in plasma corticosterone concentration. Pretreatment with prazosin did not alter the inhibitory effect of morphine...

Constant corticosterone replacement normalizes basal adrenocorticotropin (ACTH) but permits sustained ACTH hypersecretion after stress in adrenalectomized rats.

To characterize further the effects of providing a constant corticosterone signal after bilateral adrenalectomy, we have compared the effects of bilateral adrenalectomy with no replacement (ADX) and with replacement with a corticosterone pellet implanted sc at surgery (B-PELLET) to those of sham-adrenalectomy (SHAM) on pituitary and plasma ACTH concentrations during the first 3 postoperative days. In ADX rats, plasma ACTH concentrations were elevated at all times compared to those in the SHAM group; pituitary ACTH content decreased during the first 12 h, then increased and was not different from that in the SHAM group thereafter. Replacement of corticosterone at the time of adrenal surgery in B-PELLET rats resulted in no differences in pituitary and plasma ACTH concentrations from SHAM values, suggesting that immediate steroid replacement prevents the major adrenalectomy-induced changes in central regulatory components governing basal activity of the adrenocortical system. Although B-PELLET rats had normal basal morning ACTH concentrations 5 days after surgery, they exhibited augmented and sustained ACTH responses to five different ACTH-releasing stimuli (injection, restraint, chlorpromazine, and, under pentobarbital anesthesia, morphine or sham adrenalectomy). The circulating corticosterone concentrations were maintained at relatively constant, low levels (3-6 micrograms/dl). Because these concentrations appear to restore basal morning ACTH concentrations to normal, but do not restore the ACTH response to stress to normal, we conclude that a different corticosterone signal is required to normalize stress-induced ACTH responses.

Circadian variations in plasma corticosterone permit normal termination of adrenocorticotropin responses to stress.

We previously reported that adrenalectomized rats given constant corticosterone via a sc pellet (B-PELLET) hypersecrete ACTH in response to stress. Although lacking a feedback signal, B-PELLET rats do not secrete ACTH indefinitely after stress; plasma ACTH levels in these animals returned to those in sham-operated (SHAM) rats within 1-4 h after 2-min restraint. To distinguish between the requirement for circadian or stress-induced increases in corticosterone, we compared changes in ACTH and corticosterone levels after stress in SHAM and B-PELLET rats with those in cyanoketone-treated rats (CK) and adrenalectomized rats given corticosterone in their drinking fluid (B-WATER). B-WATER rats exhibited sustained increases in plasma corticosterone after lights-off, correlating with the nocturnal feeding period. Morning plasma corticosterone levels in B-WATER rats were constant and even lower than those in B-PELLET rats; however, B-WATER rats did not differ from SHAM rats in their ACTH response to ip injection. CK rats, which have an approximately normal circadian corticosterone rhythm but do not have significant corticosterone responses to acute stimuli, also exhibited plasma ACTH levels similar to those of SHAM rats at all times after 5-min restraint. Compared with SHAM and B-WATER rats in the same experiment, B-PELLET rats tended to hypersecrete ACTH 60 min after 5 min of restraint, but only had significantly elevated plasma ACTH relative to both groups 45 min after 10 min of restraint. We conclude that circadian, rather than stress-induced, increases in corticosterone may be sufficient for normal termination of ACTH responses to stress.

The suprachiasmatic nuclei stimulate evening ACTH secretion in the rat.

The effect of bilateral lesions of the suprachiasmatic nuclei (SCN) on the circadian rhythm in ACTH was studied in rats that were adrenalectomized and implanted with a subcutaneous corticosterone (B) pellet. Rats wee chronically cannulated to allow for repeated blood sampling. In rats with B pellets, bilateral lesions of the SCN eliminated the circadian rise in plasma ACTH seen in sham-lesioned animals. This is consistent with the idea that the SCN stimulate ACTH secretion in the evening.

Characterization of corticosterone feedback regulation of ACTH secretion.

Adrenalectomy-induced increases in ACTH secretion in rats are returned to normal by an action of corticosterone on the brain, not on the pituitary. Five days after adrenalectomy with constant steroid replacement, the concentration of free corticosterone in plasma which reduces plasma ACTH by 50% is approximately 0.8 nM. By contrast, the concentration of free plasma corticosterone required for 50% reduction of thymus wet weight or plasma transcortin concentration (both targets for glucocorticoid action) is about 4.5 nM. These results suggested that the inhibition of ACTH by corticosterone might be mediated by association of the steroid with high affinity, type I corticosteroid receptors, whereas the inhibition of thymus weight and transcortin might be mediated by association of the steroid with lower affinity, type II receptors. The results of studies comparing the ability of corticosterone, dexamethasone and aldosterone to inhibit adrenalectomy-induced ACTH secretion support the hypothesis that basal ACTH secretion in rats is mediated by association of corticosterone with type I receptors.

Reset of feedback in the adrenocortical system: an apparent shift in sensitivity of adrenocorticotropin to inhibition by corticosterone between morning and evening.

There is evidence in man and rats that higher circulating levels of glucocorticoids are required to normalize basal unstimulated ACTH levels at the peak of the circadian rhythm than at the trough. To explore this phenomenon, we tested the inhibitory effect of constant levels of corticosterone on plasma ACTH in the morning (AM) and evening (PM) in young male rats implanted with fused pellets of corticosterone-cholesterol at the time of adrenalectomy (ADX+B) and studied 5 days later. There was a marked shift of the plasma corticosterone-ACTH inhibition curve to the right between AM and PM, demonstrating that the efficacy of corticosterone feedback inhibition of ACTH is less in the PM. Comparison of plasma ACTH and corticosterone levels during 24 h in sham-adrenalectomized rats (SHAM-ADX), adrenalectomized rats (ADX), and ADX+B revealed constantly low ACTH in SHAM-ADX, constantly high ACTH in ADX, and biphasic ACTH levels in ADX+B. Corticosterone levels were biphasic in SHAM-ADX and were constant in the other two groups. These results again showed a shift in corticosterone feedback efficacy as a function of the time of day and also suggested that basal ACTH secretion is maintained in the low normal range in intact rats because of the marked diurnal rhythm in corticosterone. The sensitivity of the pituitary ACTH response to exogenous CRF did not change between AM and PM in either intact or ADX+B showing that the shift in feedback sensitivity to corticosterone does not reside in the pituitary. The response of the entire adrenocortical system to histamine stress was shown to be equivalent in both the AM and PM, suggesting that feedback sensitivity of the entire system to corticosterone does not change as a function of the time of day. We conclude from these results that there is an apparent diurnal change in ACTH sensitivity to corticosterone feedback that can be defined operationally as reset. We believe that the site of feedback being tested shifts solely from the pituitary in the AM (at the nadir of the rhythm) to the brain and the pituitary in the PM (at the peak of the rhythm). The lack of the normally high transients of corticosterone that occur in SHAM-ADX rats results in increased brain drive of the pituitary in ADX+B.

Effects of pargyline, reserpine and neurotoxin lesions on [3H]tryptamine binding sites in rat brain.

[3H]Tryptamine binding sites were measured in 4 areas of rat brain following treatment with either pargyline or reserpine for 12 days, or 5 days and 30 days following intraventricular injections of 6-hydroxydopamine or 5,7-dihydroxytryptamine. Pargyline treatment decreased [3H]tryptamine binding in cerebral cortex, hippocampus, striatum and hypothalamus. Reserpine treatment increased binding in the cerebral cortex and hippocampus, but not in the striatum or hypothalamus. Neither 6-hydroxydopamine nor 5,7-dihydroxytryptamine altered [3H]tryptamine binding in any of the 4 brain areas. These results indicate that [3H]tryptamine binding sites in brain may be modified by drugs that can potentially affect tryptamine metabolism, and that the sites are not located on catecholamine or serotonin axons.

Corticosterone: narrow range required for normal body and thymus weight and ACTH.

ACTH secretion appears to be under fairly tight negative feedback control by corticosteroids secreted from the adrenal cortex. In these studies we determined the circulating levels of a constant corticosterone signal that best restored body weight gain, thymus weight and ACTH levels to normal in bilaterally adrenalectomized rats given saline to drink. Young male rats were treated at the time of adrenalectomy with subcutaneously implanted pellets of wax or various ratios of corticosterone-cholesterol. Sham-adrenalectomized rats and adrenalectomized rats given corticosterone in the drinking fluid served as comparison groups. Rats were killed 3, 7, or 14 days after adrenalectomy. There was no difference in levels of plasma corticosterone in the morning and in the evening in pellet-implanted rats in contrast to the diurnal variation in the reference groups. Circulating corticosterone levels that best restored body weight, thymus weight, and resting and stress-induced ACTH levels to normal ranged between 4.5 and 7.4 micrograms/dl. Plasma corticosterone levels of 8-11 micrograms/dl were excessive and levels of 2-4 micrograms/dl were not adequate. We conclude that there is a very narrow range of plasma corticosterone compatible with normal growth rate, thymus mass and ACTH secretion. These results reveal the necessity for strict negative feedback regulation of ACTH secretion by corticosteroids.

Characterization of [3H]tryptamine binding sites in brain.

[3H]Tryptamine binds with high affinity to sites on rat brain membranes. The sites have the characteristics of tryptamine receptor recognition sites. These sites are widely distributed among rat brain regions with the highest density occurring in the cerebral cortex, striatum and hippocampus. The site is also found in human cerebral cortex. The binding site is localized mainly to the synaptosomal fraction. Drug competition studies indicate that the [3H]tryptamine binding site is distinct from serotonin receptors. Drugs that are potent inhibitors of [3H]tryptamine binding include tetrahydro-beta-carboline, quipazine, phenylethylamine, amphetamine, p-chloroamphetamine and methamphetamine.