Differential effects of intravenous R,S-(+/-)-3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and its S(+)- and R(-)-enantiomers on dopamine transmission and extracellular signal regulated kinase phosphorylation (pERK) in the rat nucleus accumbens shell and core

R,S(+/-)-3,4-methylenedioxymethamphetamine (R,S(+/-)-MDMA, 'Ecstasy') is known to stimulate dopamine (DA) transmission in the nucleus accumbens (NAc). In order to investigate the post-synaptic correlates of pre-synaptic changes in DA transmission and their relationship with MDMA enantiomers, we studied the effects of R,S(+/-)-MDMA, S(+)-MDMA, and R(-)-MDMA on extracellular DA and phosphorylated extracellular signal regulated kinase (pERK) in the NAc shell and core. Male Sprague-Dawley rats, implanted with a catheter in the femoral vein and vertical concentric dialysis probes in the NAc shell and core, were administered i.v. saline, R,S(+/-)-MDMA, S(+)-MDMA, or R(-)-MDMA. Extracellular DA was monitored by in vivo microdialysis with HPLC. Intravenous R,S(+/-)-MDMA (0.64, 1, and 2 mg/kg) increased dialysate DA, preferentially in the shell, in a dose-related manner. S(+)-MDMA exerted similar effects but at lower doses than R,S(+/-)-MDMA, while R(-)-MDMA (1 and 2 mg/kg) failed to affect dialysate DA. R,S(+/-)- and S(+)-MDMA but not R(-)-MDMA increased ERK phosphorylation (expressed as density/neuron and number of pERK-positive neurons/area) in both subdivisions of the NAc. The administration of the D1 receptor antagonist, SCH 39166, prevented the increase in pERK elicited by R,S(+/-)-MDMA and S(+)-MDMA, while the D2/3 receptor antagonist, raclopride, increased pERK in the NAc core per se but failed to affect the R,S(+/-)-MDMA-elicited stimulation of pERK. The present results provide evidence that the DA stimulant effects of racemic MDMA are accounted for by the S(+)-enantiomer and that pERK may represent a post-synaptic correlate of the stimulant effect of R,S(+/-)-MDMA on D1-dependent DA transmission.     

Evidence for a role of Hsp70 in the neuroprotection induced by heat shock pre-treatment against 3,4-methylenedioxymethamphetamine toxicity in rat brain

3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') produces acute hyperthermia which increases the severity of the selective serotoninergic neurotoxicity produced by the drug in rats. Heat shock protein 70 (Hsp70) is a major inducible cellular protein expressed in stress conditions and which is thought to exert protective functions. MDMA (12.5 mg/kg, i.p.), given to rats housed at 22 degrees C, produced an immediate hyperthermia and increased Hsp70 in frontal cortex between 3 h and 7 days after administration. MDMA, given to rats housed at low ambient temperature (4 degrees C) produced transient hypothermia followed by mild hyperthermia but no increase in Hsp70 expression, while rats treated at elevated room temperature (30 degrees C) showed enhanced hyperthermia and similar expression of Hsp70 to that seen in rats housed at 22 degrees C. Fluoxetine-induced inhibition of 5-HT release and hydroxyl radical formation did not modify MDMA-induced Hsp70 expression 3 h later. Four- or 8-day heat shock (elevation of basal rectal temperature by 1.5 degrees C for 1 h) or geldanamycin pre-treatment induced Hsp70 expression and protected against MDMA-induced serotoninergic neurotoxicity without affecting drug-induced hyperthermia. Thus, MDMA-induced Hsp70 expression depends on the drug-induced hyperthermic response and not on 5-HT release or hydroxyl radical formation and pre-induction of Hsp70 protects against the long-term serotoninergic damage produced by MDMA.     

3,4-Methylenedioxymethamphetamine increases interleukin-1beta levels and activates microglia in rat brain: studies on the relationship with acute hyperthermia and 5-HT depletion

3,4-Methylenedioxymethamphetamine (MDMA) administration to rats produces acute hyperthermia and 5-HT release. Interleukin-1beta (IL-1beta) is a pro-inflammatory pyrogen produced by activated microglia in the brain. We examined the effect of a neurotoxic dose of MDMA on IL-1beta concentration and glial activation and their relationship with acute hyperthermia and 5-HT depletion. MDMA, given to rats housed at 22 degrees C, increased IL-1beta levels in hypothalamus and cortex from 1 to 6 h and [(3)H]-(1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)3-isoquinolinecarboxamide) binding between 3 and 48 h. Increased immunoreactivity to OX-42 was also detected. Rats became hyperthermic immediately after MDMA and up to at least 12 h later. The IL-1 receptor antagonist did not modify MDMA-induced hyperthermia indicating that IL-1beta release is a consequence, not the cause, of the rise in body temperature. When MDMA was given to rats housed at 4 degrees C, hyperthermia was abolished and the IL-1beta increase significantly reduced. The MDMA-induced acute 5-HT depletion was prevented by fluoxetine coadministration but the IL-1beta increase and hyperthermia were unaffected. Therefore, the rise in IL-1beta is not related to the acute 5-HT release but is linked to the hyperthermia. Contrary to IL-1beta levels, microglial activation is not significantly modified when hyperthermia is prevented, suggesting that it might be a process not dependent on the hyperthermic response induced by MDMA.     

Studies,using in vivo microdialysis,on the effect of the dopamine uptake inhibitor GBR 12909 on 3,4-methylenedioxymethamphetamine ('ecstasy')-induced dopamine release and free radical formation in the mouse striatum

The present study examined the mechanisms by which 3,4-methylenedioxymethamphetamine (MDMA) produces long-term neurotoxicity of striatal dopamine neurones in mice and the protective action of the dopamine uptake inhibitor GBR 12909. MDMA (30 mg/kg, i.p.), given three times at 3-h intervals, produced a rapid increase in striatal dopamine release measured by in vivo microdialysis (maximum increase to 380 +/- 64% of baseline). This increase was enhanced to 576 +/- 109% of baseline by GBR 12909 (10 mg/kg, i.p.) administered 30 min before each dose of MDMA, supporting the contention that MDMA enters the terminal by diffusion and not via the dopamine uptake site. This, in addition to the fact that perfusion of the probe with a low Ca(2+) medium inhibited the MDMA-induced increase in extracellular dopamine, indicates that the neurotransmitter may be released by a Ca(2+) -dependent mechanism not related to the dopamine transporter. MDMA (30 mg/kg x 3) increased the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) from salicylic acid perfused through a probe implanted in the striatum, indicating that MDMA increased free radical formation. GBR 12909 pre-treatment attenuated the MDMA-induced increase in 2,3-DHBA formation by approximately 50%, but had no significant intrinsic radical trapping activity. MDMA administration increased lipid peroxidation in striatal synaptosomes, an effect reduced by approximately 60% by GBR 12909 pre-treatment. GBR 12909 did not modify the MDMA-induced changes in body temperature. These data suggest that MDMA-induced toxicity of dopamine neurones in mice results from free radical formation which in turn induces an oxidative stress process. The data also indicate that the free radical formation is probably not associated with the MDMA-induced dopamine release and that MDMA does not induce dopamine release via an action at the dopamine transporter.     

Short- and long-term effects of vinblastine on the rat adrenal medulla

Summary The effects of a single high dose (10mg/kg) of vinblastine (vb) sulfate (Velbe, Lilly) on the ultrastructure, catecholamine (CA) content and activity of CA-synthesizing enzymes of the rat adrenal medulla were studied for up to 120h after intravenous injection of the drug.By 1 h, microtubules were virtually absent from chromaffin cells and preganglionic cholinergic axons, and typical paracrystals had appeared inside the nerve fibers. By 16h microtubules were completely reconstituted and paracrystals had disappeared. From 16h onwards, there was an increasing depletion of storage granules from adrenaline (A) — producing cells, which coincided with biochemical determinations showing a reduction of adrenal A to about 40 % of control levels by 48 h, with noradrenaline (NA) remaining in the range of controls. Both A- and NA-storing cells showed an extensive proliferation of the rough endoplasmic reticulum (ER). Vb caused a marked increase in tyrosine hydroxylase (TH; +113%) and dopamine -hydroxylase (DBH; +82%) activities after 48 h. Splanchnicotomy completely abolished the vb-mediated increase in TH and DBH activities. A smaller increase (+ 47 %) in enzyme activity was observed with phenylethanolamine N-methyltransferase (PNMT). Vb (10^–5M) had no apparent effect on granule content and the amount of rough ER in chromaffin cells, which were cultured for 48 h.The results demonstrate that a single high dose of vb has relatively little short-term effects on the rat adrenal medulla, but causes drastic long-term changes in CA-content and enzyme activities that are mediated by the preganglionic nerves. These changes could be interpreted as an effort to compensate for a loss of CA-stores in peripheral adrenergic nerves (cf. Cheney et al., 1973). The differential long-term effect of vb on adrenal NA and A might be due to the lower induction of PNMT as compared to TH and DBH activities and/or to a preferential release of A versus NA, which may occur at high frequencies of stimulation of the splanchnic nerves.Supported by grants from the Deutsche ForschungsgemeinschaftDedicated to Professor G. Petry in honor of his 65th birthday     

Elevated corticosterone levels in stomach milk,serum, and brain of male and female offspring after maternal corticosterone treatment in the rat

Early influences such as maternal stress affect the developmental outcome of the offspring. We created an animal model of postpartum depression/stress based on giving high levels of corticosterone (CORT) to the rat dam, which resulted in behavioral and neural changes in the offspring. This study investigated whether highly elevated levels of maternal CORT during pregnancy or the postpartum result in higher levels of CORT in the stomach milk, serum, and brain of offspring. Dams received daily injections of CORT (40 mg/kg) or oil (control) either during pregnancy (gestational days 10–20) or the postpartum (Days 2–21). Pups that were exposed to high gestational maternal CORT had higher CORT levels in serum, but not in stomach milk or brain, on postnatal day (PND) 1. However, on PND7, pups that were exposed to high postpartum maternal CORT had higher CORT levels in stomach milk and brain, but not in serum. Conversely on PND18, pups that were exposed to high postpartum maternal CORT had higher CORT levels in serum, but not in brain (prefrontal cortex, hypothalamus, or hippocampus). Moreover, 24 h after weaning, there were no significant differences in serum CORT levels between the groups. Thus, CORT given to the dam during pregnancy or the postpartum results in elevated levels of CORT in the offspring, but in an age‐ and tissue‐dependent manner. Developmental exposure to high CORT could reprogram the HPA axis and contribute to the behavioral and neural changes seen in adult offspring. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 714–725, 2010     

Short- and long-term effects of ACTH on the adrenal zona glomerulosa of the rat

Summary Short-term ACTH treatment provoked a decrease in volume of the lipid-droplet compartment in rat zona glomerulosa cells, and a rise in plasma and intracellular concentrations of corticosterone and aldosterone. It enhanced activities of 3-hydroxysteroid dehydrogenase (3HSD), 11-hydroxylase (11OH) and 18-hydroxylase (18OH). Long-term ACTH administration produced a hypertrophy of the zona glomerulosa and its parenchymal cells, a result of the increase in volume of the smooth endoplasmic reticulum and the mitochondrial compartment. The surface area per cell of mitochondrial inner membranes increased; the tubular cristae were transformed into a homogeneous population of vesicles. The plasma and intracellular concentrations of corticosterone further increased, whereas those of aldosterone fell below basal levels (the aldosterone-escape phenomenon). The activities of 3HSD and 11OH were enhanced, that of 180H decreased. Therefore, ACTH stimulates zona glomerulosa growth and transforms parenchymal elements into zona fasciculata celltypes. Cyanoketone nullified acute ACTH effects on plasma and intracellular concentrations of corticosterone and aldosterone, but did not affect the activities of 11OH and 18OH. Chronic ACTH treatment produced similar results, although 18OH activity was not suppressed. The mechanism underlying the aldosterone-escape phenomenon may thus involve a rise in the intracellular concentration of corticosterone, caused by the enhanced synthesis and activation of 3HSD and 11OH.     

Effects of deprenyl on monoamine oxidase and neurotransmitters in the brains of MPTP-treated aging mice

Deprenyl is a selective monoamine oxidase B (MAO-B) inhibitor and has been used in the treatment of Parkinson's disease. However, it is not known whether deprenyl effects are symptomatic or pharmacological. Aging mice were partially lesioned with MPTP. Control and MPTP-treated mice were given deprenyl in drinking water for 14 days. Brain tissue (including the striatum, olfactory tubercle and cerebral cortex) was assayed for MAO-B and neurotransmitter levels. The results show that deprenyl treatment, given alone or after MPTP, reduced MAO-B activity in all the three regions. No change was seen in dopamine (DA), 3,4-dihydroxyphenyl acetic acid (DO-PAC), and homovanillic acid (HVA) content in any of the three areas. Cortical norepinephrine (NE) levels were also unaltered. However, striatal serotonin (5-HT) levels were decreased while its metabolite, 5-HIAA levels were significantly increased in the olfactory tubercle in animals receiving deprenyl alone. These data suggest that deprenyl treatment reduces MAO-B activity in regions in addition to the striatum without affecting norepinephrine, dopamine (DA) and its metabolites.     

Hypothalamic metabolism of neurotransmitters (serotonin,norepinephrine, dopamine) and NPY,and gonadal and adrenal activities,during the early postnatal period in the rat

It is noteworthy that in the rat the early postnatal life is marked by an activation of both the corticostimulating function of the adenohypophysis in neonates of both sexes and of the gonadostimulating function mainly in males. In order to specify if such neuroendocrine variations are temporally correlated with changes in the hypothalamic metabolism of neurotransmitters, the hypothalamic metabolism of serotonin (5 HT), norepinephrine (NE), and dopamine (DA) and the hypothalamic content of neuropeptide Y (NPY) have been investigated in newborn rats of both sexes, delivered at term by cesarean section, as well as changes in the activity of both the hypothalamo-pituitary adrenal axis (HPA) and the hypothalamo-pituitary gonadal axis (HPG). Experimental data suggested that 1) in males a rise in hypothalamic metabolism of 5 HT, NE and DA occurs during the first two hours after delivery, whereas in females, only the metabolism of NE increases. Moreover, the postnatal metabolism of NE was higher in females than in littermate males; 2) NPY content of the hypothalamus, which was at birth significantly higher in males than in females, dropped in the former but not in the latter; 3) in newborn males, an early surge of plasma testosterone occurs, suggesting postnatal activation of the HPG axis; on the other hand, in females, a late and slight increase in plasma estradiol is observed; 4) in early postnatal life, a sex-independent rise in plasma ACTH and adrenal and plasma corticosterone levels suggest a comparable activation of the HPA axis in newborns of both sexes. In conclusion, the early postnatal activation of the corticostimulating function in neonates of both sexes and that of the gonadostimulating function, mainly in males, could be temporally correlated with a rise in the hypothalamic metabolism of two neurotransmitters, 5 HT and NE, and of NPY content. According to our data, a sex-dependent metabolsim of neurotransmitters in the hypothalamus is already apparent in early postnatal life.     

Short- and long-term effects of biliary drainage on hepatic cholesterol metabolism in the rat.

The present study concerns short- and long-term effects of interruption of the enterohepatic circulation (EHC) on hepatic cholesterol metabolism and biliary secretion in rats. For this purpose, we employed a technique that allows reversible interruption of the EHC, during normal feeding conditions, and excludes effects of anaesthesia and surgical trauma. [3H]Cholesteryl oleate-labelled human low-density lipoprotein (LDL) was injected intravenously in rats with (1) chronically (8 days) interrupted EHC, (2) interrupted EHC at the time of LDL injection and (3) intact EHC. During the first 3 h after interruption of the EHC, bile flow decreased to 50% and biliary bile acid, phospholipid and cholesterol secretion to 5%, 11% and 19% of their initial values respectively. After 8 days of bile diversion, biliary cholesterol output and bile flow were at that same level, but bile acid output was increased 2-3-fold and phospholipid output was about 2 times lower. The total amount of cholesterol in the liver decreased after interruption of the EHC, which was mainly due to a decrease in the amount of cholesteryl ester. Plasma disappearance of LDL was not affected by interruption of the EHC. Biliary secretion of LDL-derived radioactivity occurred 2-4 times faster in chronically interrupted rats as compared with the excretion immediately after interruption of the EHC. Radioactivity was mainly in the form of bile acids under both conditions. This study demonstrates the very rapid changes that occur in cholesterol metabolism and biliary lipid composition after interruption of the EHC. These changes must be taken into account in studies concerning hepatic metabolism of lipoprotein cholesterol and subsequent secretion into bile.     

Effect of stress and dexamethasone treatment on circadian rhythms of melatonin and corticosterone in ring dove (Streptopelia risoria)

The possible relationship between the circadian rhythm of blood levels of melatonin and corticosterone in ring dove (Streptopelia risoria) subjected to both immobilization stress and immobilization stress plus dexamethasone treatment were studied. The results show changes in the circadian rhythm of melatonin, with increased day-time levels in situations of stress accompanied by increased corticosterone levels. The highest blood melatonin levels over the 24 h of the study were obtained when the animals were treated with dexamethasone and then subjected to stress. Given the antioxidant role of melatonin, our results support the idea of melatonin-corticosterone coupling with the possibility that melatonin released in situations of stress counteracts the adverse effects of glucocorticoids on the organism.     

Determination of 3,4-methylenedioxymethamphetamine and its five main metabolites in rat urine by solid-phase extraction and high performance liquid chromatography with on line mass spectrometry

The consumption of psychostimulant amphetamine-like drugs has increased significantly in recent years. Some MDMA metabolites are probably involved in the neurotoxicity and neurodegeneration caused by prolonged use rather than MDMA itself. We recently developed a method to analyze MDMA and its five main metabolites in rat plasma [7]. We have now fully validated this method to the quantification of these drugs in rat urine. We extracted MDMA and its metabolites with Oasis WCX cartridges, separated them on a Nucleodur C18 analytical column and quantified them by ion-trap mass spectrometry. Linearity was excellent: 12.5–1250 ng/mL urine for HMA, HMMA, MDA and MDMA, 25–2500 ng/mL for HHMA, and 150–7500 ng/mL for HHA (r² > 0.993 for all analytes). The lower limits of quantification were 12.5 ng/mL urine for MDMA, MDA, HMA and HMMA, 25 ng/mL for HHMA and 150 ng/mL for HHA. Reproducibility was good (intra-assay precision = 1.7–6.1%; inter-assay precision = 0.6–5.7%), as was accuracy (intra-assay deviation = 0.1–4.8%; inter-assay deviation = 0.7–7.9%). Average recoveries were around 85.0%, except for HHMA (66.2%) and HHA (53.0%) (CV < 8.3%). We also checked the stability of stock solutions and the internal standards after freeze-thawing and in the autosampler. Lastly, we measured the MDMA, MDA, HHMA, HHA, HMMA and HMA in urine samples taken over 24 h from rats given subcutaneous MDMA.     

The anticonvulsant effect of citalopram on El mice,and the levels of tryptophan and tyrosine and their metabolites in the brain

Serotonin (5-HT) plays an important role in the seizures of El mice since the seizure threshold of El mice correlates with the 5-HT concentration in the central nervous system. In this study, the anticonvulsant effect of a 5-HT reuptake blocker, citalopram, was evaluated behaviorally and biochemically. El mouse convulsions were inhibited by oral administration of citalopram for 2 weeks. Citalopram increased tryptophan and tyrosine amounts, and decreased the 5-HT, 5-hydroxy-indoleacetic acid, kynurenine, and dopamine amounts in the brain. These findings show that citalopram depresses monoaminergic metabolism. Given the known convulsant effect of kynurenine, it is suggested that its decrease by citalopram may involve attenuation of El mice seizures.     

Short- and long-term effects of high milking frequency during the first 21 days of lactation on production and reproductive performance in high-lactating cows

We examined short- and long-term effects of high milking frequency (HMF) for the first 21 days of lactation. The study included 122 Israeli Holstein cows – 32 pregnant heifers, 40 cows in second lactation and 50 cows in >second lactation. Heifers were paired according to predicted transmitting ability and cows according to energy-corrected milk (ECM) production, age, days in milk and expected calving date. Thin cows (body condition score <2.75) were not included. One cow from each pair was arbitrarily allocated to a control group milked three times daily (3× milking cows) and the counterpart to an experimental group milked six times daily for the first 21 days of lactation and then three times daily for the rest of the lactation (6× milking cows). During the first 21 days of lactation, 6× milking cows produced 9.3 kg more milk (26.5%) and 7.16 kg more ECM (19%) than the 3× milking cows. The higher milk production persisted throughout the entire lactation (305 days), as reflected by treatment×age interaction showing higher milk production for the first and second (7%) but not >second lactation cows relative to their control counterparts (−0.37%); ECM production was also higher in 6× milking first and second lactation (7.6% and 5%, respectively) but not for >second lactation cows. Furthermore, HMF had long-lasting effects, expressed as significantly higher milk production through the succeeding lactation in the previous first lactation cows (10%); a tendency toward significance in the second lactation cows relative to the controls (4.7%), but a deleterious effect on the >second lactation cows, reflected by lower milk production (−5.25%) than in controls; similar patterns were found for the ECM. For the entire 305 days of lactation, fat and protein yields were higher for first and second lactation cows, whereas protein yield for >second lactation cows was lower in the 6× milking v. control group. Given that HMF during the first 21 days of first or second lactation increases milk and ECM yields throughout the concurrent and successive lactation with no adverse effect on energy balance, mastitis, metabolic diseases or reproduction, it seems to be economically beneficial. However, caution should be paid for >second lactation cows due to absence of significant effect in the entire of the first HMF applied lactation and the deleterious effect in the succeeding lactation.     

5-HT和DA对虾蟹打斗行为影响的研究进展

5-羟色胺(5-HT)和多巴胺(DA)是两种神经递质,可与众多不同类型的受体结合发挥多种重要的生理功能.现已证明其广泛分布于多种动物的不同组织中,在动物的打斗行为活动中起着重要的调节作用.目前,在虾蟹中已被报道的5-HT受体主要有5种,分别是5-HT1A、5-HT1B、5HT2A、5HT2B和5-HT7;DA受体主要为DA1A、DA1B、DA2和DA4.5-HT和DA及其受体分布存在明显的种属和组织特异性.5-HT和DA参于了虾蟹打斗行为的调节过程并有不同的调节机理.5-HT可以调节环磷酸腺苷(cAMP)或高血糖激素(CHH)的释放,促进或抑制虾蟹打斗行为;而DA同样能够通过调节cAMP及COMT等物质的释放来调节虾蟹打斗行为.     

Fetal and neonatal rat pancreas in organ culture: age-related effects of corticosterone on the development of the islet cells

Fetal (18 days postcoitum) and neonatal (3-day) pancreatic explants were grown in organ culture with or without supplementation with corticosterone (0.1 micrograms/ml). After 0, 4, and 8 days of culture, the specific hormone-positive, islet cell volumes were determined by the use of immunocytochemical and morphometric methods. The insulin, glucagon, and somatostatin contents of the explants were estimated by radioimmunoassays. In the fetal explants, all of the islet cell populations increased in volume and the content of each of the hormones increased over an 8-day period of culture. Supplementation with corticosterone resulted in a restriction of the increases of the alpha and delta cell volumes and in the somatostatin content of the explants. In the neonatal explants, the volumes of the alpha and delta cells and the glucagon and somatostatin contents decreased over a 4-day culture period. The presence of corticosterone in the culture medium preserved these cells and their hormone content. Co-culture of 18-day fetal and 3-day neonatal pancreata in control medium for 8 days resulted in a significant decrease in the content of all three of the islet hormones in the fetal explants. These results suggest that a substance harmful to the islet cells is released from the degenerating acinar cells. Thus, the effects of the steroid on the islets may be mediated through its effects on the acinar tissue.     

Effect of endogenous corticosterone on the determination of dexamethasone receptor levels in rat liver cytosol

The effect of endogenous corticosterone on the quantitative measurement of dexamethasone receptors in liver cytosols from developing rats has been studied. Liver cytosols from adrenalectomized rats were preincubated with increasing concentrations of nonlabeled corticosterone and the levels of detectable dexamethasone receptors were subsequently determined either directly or after removal of unbound corticosterone. Corticosterone concentrations of 50 nM or lower had no significant effect on the specific binding of labeled dexamethasone. Higher concentrations of corticosterone resulted in under-estimation of dexamethasone receptor levels. The mean levels of endogenous corticosterone in liver cytosols from 19.5- to 21.5- day fetuses, 22-day fetuses, 6-day-old immature rats and adult rats were 27.40, 11.91, 0.81 and 4.05 nM, respectively. It is concluded that variations in the levels of circulating corticosterone in the rat under normal physiological conditions have no significant effect on the quantitative measurement of total (occupied and unoccupied) receptor sites for dexamethasone in liver cytosol. This is supported by the finding that prior treatment of liver cytosols, from rats at different stages of development, with charcoal to remove unbound steroids has no effect on the amount of detectable dexamethasone receptors.