Stress mediated changes in hypothalamic corticotropin releasing factor-like immunoreactivity

Hypothalamic corticotropin releasing factor-like immunoreactivity (CRF-LI), plasma ACTH and corticosterone levels were measured by radioimmunoassay over a two hour period of restraint stress. The results of this study demonstrate a significant decrease in hypothalamic CRF-LI levels 15 and 30 minutes after the start of restraint stress which is followed by a significant increase at 60 minutes that is abolished by cycloheximide pretreatment. Plasma ACTH and corticosterone levels were significantly elevated after 15, 30, 60, 90, and 120 minutes of restraint stress. These results are consistent with a release of CRF from the hypothalamus during stress. The cycloheximide-sensitive increase in hypothalamic CRF-LI indicates that synthesis of CRF-41 occurs during prolonged stress. These results suggest that the response of an organism to exposure to a long-term, high intensity stress involves both the release and synthesis of CRF-41.     

Cocaine induced secretion of ACTH, beta-endorphin, and corticosterone

The effect of intraperitoneal administration of cocaine on the concentrations of hypothalamic corticotropin releasing factor like-immunoreactivity (CRF-LI), plasma ACTH, beta-endorphin, and corticosterone was investigated. Groups of rats were injected with 20 mg/kg cocaine HCI or 0.9% NaCl and then killed 0, 10, 20, 30 or 60 minutes later. Hypothalamic CRF-LI, plasma ACTH, beta-endorphin, and corticosterone concentrations were determined by radioimmunoassay. A significant increase in plasma ACTH, beta-endorphin, and corticosterone concentrations was observed after cocaine administration. In contrast, cocaine had no significant effect on hypothalamic CRF-LI concentrations. Intravenous administration of 0.5 and 2.0 mg/kg cocaine to rats in which the endogenous release of CRF was blocked by chlorpromazine, morphine, and pentobarbital elicited a significant increase in plasma corticosterone concentrations. These results demonstrate that cocaine induces the release of ACTH, beta-endorphin, and corticosterone and suggest that this response is mediated at the pituitary level.     

Immunoneutralization of corticotropin-releasing hormone prevents the diurnal surge of ACTH

To determine whether CRH is required for the evening rise in plasma ACTH, rats were injected at 0800 hr with CRH antiserum (anti-CRH) or normal rabbit serum (NRS). Blood samples were taken through venous catheters at 0800 hr before treatment and at 1300, 1700, and 2100 hr. Plasma was assayed for immunoreactive ACTH and corticosterone. There was no significant difference in pretreatment values between the two groups. Immunoneutralization of CRH abolished the rise in plasma ACTH seen at 1700 hr in the NRS group but had little effect on earlier levels. The diurnal elevation in plasma corticosterone continued after anti-CRH treatment, but peak levels occurred earlier. Plasma ACTH and corticosterone were significantly correlated at the time of the diurnal surge, but not at 0800 hr or 1300 hr in the NRS controls or at any time point in the anti-CRH group. These results suggest that CRH is required for the diurnal surge of plasma ACTH. They also confirm previous observations by others that the adrenal cortex does not require active CRH or a diurnal surge of ACTH in order to exhibit a significant diurnal increase in secretion of corticosterone, and that factors other than CRH may be relatively more active than CRH in regulation of ACTH secretion during the time of circadian inactivity.     

Hypothalamic and pituitary periodicity demonstrated by isolated rat adrenal cells

Rats were maintained under standardized conditions of food and water ad libitum and a lighting schedule of 12 h light/we h dark for seven days. Animals were sacrificed at 0500 and 1700 h and tissues were removed and washed. Isolated intact adrenal and pituitary cells were prepared by collagenase treatment. Using a sequential incubation procedure, the release of pituitary ACTH by hypothalamic acid extracts (CRF) was assayed by stimulation of corticosteroid secretion from isolated adrenal cells. Maximum stimulation of adrenal steroids was achieved with hypothalamic extracts and pituitary cells from rats sacrificed at 1700 h, and minimum values were obtained at 0500 h. Intermediate levels were obtained when hypothalamic extracts at one time point were incubated with pituitary cells at the other. The results substantiate the late afternoon peak level of hypothalamic and pituitary secretion occurring prior to the peak activity pattern of the rat.     

Vasopressin pressor receptor-mediated activation of HPA axis by acute ethanol stress in rats

The plasma arginine vasopressin (AVP), ACTH, and corticosterone levels and the hypothalamic corticotropin-releasing hormone (CRH) content were measured after oral administration of 1 ml of 75% ethanol to rats, a model known to induce acute gastric erosions and stress. Elevated plasma AVP, ACTH, and corticosterone levels were detected 1 h after ethanol administration. Treatment with the vasopressin pressor (V(1)) receptor antagonist [d(CH(2))(5)Tyr(Me)-AVP] before ethanol administration significantly reduced the ACTH and corticosterone level increases. A higher hypothalamic CRH content was measured at 30 or 60 min after ethanol administration. V(1) receptor antagonist injection, 5 min before ethanol administration, inhibited the rise in hypothalamic CRH content. The protein synthesis blocker cycloheximide prevented the hypothalamic CRH content elevation after stress. The AVP-, CRH-, and AVP + CRH-induced in vitro ACTH release in normal anterior pituitary tissue cultures was also prevented by pretreatment with the V(1) receptor antagonist. The results support the hypothesis that stress-induced AVP may not only act directly on the ACTH producing anterior pituitary cells but also indirectly at the hypothalamic level via the synthesis and release of CRH.     

Repercussions of anterior hypothalamic lesions on nycthemeral variations of plasma thyroxine and corticosterone in the pigeon

Plasma thyroxine and corticosterone levels were determined by competitive protein binding assay, at 3 hr intervals, throughout the photoperiod. Pigeons were kept in controlled environment (21 +/- 1 degree C; 14L6-20: 10D). Intact controls exhibited low thyroxine (T4) and corticosterone (B) levels for the light phase of the photoperiod. Values were rising during the night, up to a peak at 03 hr. Electrolytic lesions were placed bilaterally in either the nucleus anterior medialis hypothalami or the n. preopticus, or the n. supraopticus. Circadian rhythms of both T4 and B were markedly altered in all lesioned pigeons, with a shift of very high T4 values to the morning times and a complete disorganization of B patterns, with very heterogeneous values. The possibility is raised that anterior hypothalamic formations participate in the endogenous oscillator circuitry in birds.     

Orcadian Changes in Stimulus Threshold and in the Effect of a Local Anaesthetic Drug in Human Teeth: Studies with an Electronic Pulptester

An electronic puiptester with constant testing current was used to study the stimulus threshold in human teeth of 28 healthy volunteers and the duration of local anaesthesia by articaine plus epinephrine in 55 patients with caries who underwent a filling therapy. In 21 out of the 28 volunteers, significant daily variations in stimulus threshold were detected by cosinor analysis. Acrophases, however, were scattered over the 24-hr scale. In patients, the local anaesthetic effect was studied at four different times of day (0800,1100,1400 and 1700 hr). The electronic device allowed one to accurately determine the time to peak effect (T_max), duration of effect (E_max, time to return to baseline threshold (T_ret) and the area under the time-effect curve (AUC) as a measure of the total local anaesthetic effect. Significant diurnal variations in AUC and E_max were found in 36 patients with the 0.8 ml dose, with peak and trough values at 1400 and 1700 hr, respectively. No differences in effect were found with the low dose of 0.4 ml applied to 19 patients either at 1400 or 1700 hr giving evidence for a circadian phase-dependency in the dose-response relationship of a local anaesthetic drug. The results clearly demonstrate that this electronic device is suitable for exact evaluation of circadian changes in local anaesthetic effects. Under drug-free control conditions, however, the low stimulus frequence of 5 Hz of this device obviously does not allow to discriminate between stimuli modified by pain perception and/or alertness.     

Radioimmunoassay of CRF-like material in rat plasma: validation of the method

The availability of antibodies against the ovine corticotropin releasing factor (CRF), which cross-react with a CRF-like immunoreactivity (CRF-LI) in the rat, has enabled us to develop a radioimmunoassay (RIA) for rat CRF-LI in plasma and crude hypothalamic extracts. 125I-Tyr CRF 1-41 was used as the tracer, and synthetic ovine CRF as the reference hormone. The precision profile of the assay indicates a high degree of reproducibility except for the lower dose range. The minimum detectable dose was 20 pg/tube. This assay can detect differences in plasma CRF-LI levels after various manipulations that simultaneously alter the ACTH levels in plasma. A wide range of CRF concentrations has been found in plasma of normal rats. Caution should be exercised in the interpretation of the values obtained since an ovine RIA system was used.     

Changes in hypothalamic LH-RH content and blood levels of LH-RH, gonadotropin and estradiol during the preovulatory stage of rat estrous cycle.

In order to investigate the sequence of events concerning gonadotropin surge, serum LH, FSH and estradiol concentrations were measured during the rat estrous cycle as well as hypothalamic and blood levels of LH-RH in the preovulatory stage. Normally cyclic female Wistar rats kept on 12 hr light (from 22.00 hr to 10.00 hr) and 12 hr dark were killed at different times of day during each stage of the cycle. The hypothalamus was quickly dissected out, divided into 3 portions (the anterior, middle and posterior) and extracted in 90% methanol. Blood LH-RH was extracted by affinity chromatography prior to radioimmunoassay. The content of LH-RH in the anterior and middle hypothalamus started to decrease between 1.00 hr-3.00 hr, reached its nadir at 6.00 hr on proestrus and recovered to its previous values on estrus. Almost simultaneously blood LH-RH concentration showed an increase of 18.3 pg/ml-8.8 pg/ml between 1.00 hr-3.00 hr, and then fell to less than 1.0 pg/ml at 6.00 hr. On the other hand, serum estradiol level began to elevate on diestrus II followed by its peak at 6.00 hr on proestrus, while the peaks of serum LH and FSH were observed at 8.00 hr and 10.00 hr, respectively. These studies indicate that the elevation of serum estradiol was followed by the release of LH-RH from the hypothalamus and the LH-RH may be responsible for the preovulatory discharge of gonadotropin.     

Excretion of corticosteroid metabolites in urine and faeces of rats.

Stress enhances the production of corticosteroids by the adrenal cortex, resulting in the increased excretion of their metabolites in urine and faeces. An intraperitoneal injection of radioactive corticosterone was applied to adult, male Sprague-Dawley rats to monitor the route and delay of excreted metabolites in urine and faeces. Peak concentrations appeared in urine after 3.2 +/- 1.9 h and in faeces after 16.7 +/- 4.3 h. Altogether about 20% of the recovered metabolites were found in urine and about 80% in faeces. Using high-performance liquid chromatography (HPLC), several peaks of radioactive metabolites were found. Some metabolites were detected by enzyme immunoassay (EIA) using two different antibodies (corticosterone, 11beta-OH-aetiocholanolone). There was a marked diurnal variation with low levels of faecal corticosterone metabolites in the evening and higher values in the morning. This diurnal variation was influenced neither by the intraperitoneal injection of isotonic saline nor by ACTH. However, the administration of dexamethasone eliminated the morning peak for 2 days.     

Involvement of hypothalamic somatostatin in glucagon-induced suppression of growth hormone secretion in conscious rats

The role of central glucagon in regulating GH secretion was studied in conscious male rats with chronic indwelling intra-atrial and intracerebro-ventricular (ICV) cannulae. Repeated blood sampling every 20 min from 1000 hr to 1700 hr showed two major GH bursts occurring at regular intervals (3.6±0.1 hr) around 1200 hr and 1540 hr. The ICV (lateral ventricle) injection of glucagon (10 μg/rat) at 1100 hr inhibited spontaneous GH secretion, and the mean (±SE) plasma GH levels from 1120 hr to 1700 hr were lower than those in controls injected ICV with the vehicle solution only (31.9±7.8 ng/ml vs. 157.1±13.4 ng/ml, p<0.01). The GH bursts did not appear until 5 hr after the injection. The intravenous (IV) injection of glucagon (10 μg/rat) did not change plasma GH levels or the occurrence of spontaneous GH bursts. The glucagon-induced suppression of GH release was attenuated when anti-somatostatin serum (ASS), but not normal rabbit serum (NRS), was given IV in a volume of 0.25 ml immediately before the ICV injection of glucagon (10 μg/rat) (mean GH levels at 1120–1700 hr: ASS+glucagon, 133.6±26.7 ng/ml vs. NRS+glucagon, 30.5±7.4 ng/ml, p<0.01). These findings suggest that central glucagon may play an inhibitory role in regulating GH secretion by stimulating SRIF release from the hypothalamus in the rat.     

Circadian variation of DSIP-like material in rat plasma

Levels of delta-sleep-inducing peptide-like immunoreactivity (DSIP-LI) in rat plasma were measured by radioimmunoassay and found to exhibit a circadian rhythm that parallelled the normal rhythm for corticosterone. The maximal plasma levels of both substances were observed to occur at about 1700h. The lowest concentrations of DSIP-LI and corticosterone were detected at 2400h and 1000h, respectively. Exposure to constant levels of illumination abolished the rhythm of DSIP-LI. It is possible that the temporal parallelism between the levels of DSIP-LI and corticosterone may represent a functional relationship between both compounds.     

Effects of U-50,488H and U-50,488H withdrawal on catecholaminergic neurons of the rat hypothalamus

Previous report from our laboratory showed that morphine produces a stimulatory effect of hypothalamic noradrenaline (NA) turnover concurrently with enhanced pituitary-adrenal response after its acute injection and during withdrawal. In the present work we have studied the effects of acute and chronic administration of the kappa agonist U-50,488H as well as the influence of U-50,488H withdrawal on the activity of hypothalamic NA and dopamine (DA) neurons and on the activity of hypothalamic-pituitary-adrenal (HPA) axis. A single dose of U-50,488H (15 mg/kg i.p.) significantly increased hypothalamic NA and decreased DA turnover at the time of an enhanced corticosterone release. Rats rendered tolerant to the kappa agonist by administration of U-50,488H twice a day for 4 days showed no changes in corticosterone secretion. Additionally, a decrease in both hypothalamic MHPG (the cerebral NA metabolite) production and NA turnover was observed, whereas DOPAC concentration and DA turnover were enhanced, which indicate the development of tolerance towards the neuronal and endocrine actions of U-50,488H. After naloxone (3 mg/kg s.c.) administration to U-50,488H-tolerant rats, we found neither behavioural signs of physical dependence nor changes in hypothalamic catecholaminergic neurotransmission. In addition, corticosterone secretion was not altered in U-50,488H withdrawn rats. Present data clearly indicate that tolerance develops towards the NA turnover accelerating and DA turnover decreasing effect of U-50,488H. Importantly and by contrast to mu agonists, present results demonstrate that U-50,488H withdrawal produce no changes in hypothalamic catecholamines turnover or in corticosterone release (an index of the hypothalamus-pituitary-adrenal activity), which indicate the absence of neuroendocrine dependence on the kappa agonist. As has been proposed, this would suggest that the mu and the kappa receptor be regulated through different cellular mechanisms, as kappa agonists have a lower proclivity to induce dependence.     

Diurnal rhythm of galanin-like immunoreactivity in the paraventricular and suprachiasmatic nuclei and other hypothalamic areas

The peptide galanin (GAL), when injected into the rat hypothalamus, is known to stimulate feeding behavior and affect the secretion of various hormones, including insulin and the adrenal steroid, corticosterone. To determine whether endogenous peptide levels shift in relation to natural rhythms of feeding and circulating hormone levels, rats were sacrificed at different times of the light/dark cycle, and their GAL levels were measured, via radioimmunoassay, in medial hypothalamic dissections and micropunched hypothalamic areas. The results suggest the existence of two distinct diurnal rhythms for hypothalamic GAL. One rhythm, detected exclusively in the area of the SCN, is characterized by bimodal peaks of GAL, threefold higher than basal peptide levels, around the onset of the dark and light periods. The second rhythm shows a single peak of GAL towards the middle of the nocturnal feeding cycle, specifically between the third and sixth hour. This latter rhythm is evident in the dorsal region of the medial hypothalamus, localized specifically to the lateral portion of the PVN. Moreover, it is inversely related to circulating insulin but unrelated to the adrenal steroids, suggesting a possible association between this pancreatic hormone and GAL in the PVN.     

Effect of muscimol (GABA-agonist) on thalamo-hypothalamic regulation by corticotropic efferents (thalamic model)

It can be assumed from previous data that the stress-induced polyphasic adrenocortical response involves two phenomenons. First, direct hypothalamic afferents stimulate the adrenocorticotropic axis resulting in a rapid increase in plasma corticosterone levels up to a maximum of about 40 ng . ml-1 at 15 min. Then, a thalamic-hypothalamic loop generates the late rebounding components. Intravenous injection of muscimol (0.5 ml . kg-1) produces only slight and short duration (45 min) disturbances (piloerection; panting; corticosteronemia). Stress application, 1 hr after muscimol administration, elicits only the first peak of corticosterone with magnitude and delay similar to that in control. However, the late rebounding component is no more visible. Thus, GABA-ergic stimulation leads to a situation similar to that seen after section of neural connections between thalamic nuclei and infundibular complex. The hypothalamic-adrenocorticotropic reactivity itself is not affected whereas the function of the regulating thalamo-hypothalamic loop is markedly impaired.     

Site of action of 5-hydroxytryptophan and 5-hydroxytryptamine on the hypothalamo-pituitary-adrenal system in vitro.

5-hydroxytryptamine at a concentration of 0.04 microgram/ml was able to block the ACTH release caused by hypothalamic tissue (CRF) while it was ineffective when a hypothalamic extract containing CRF was used. 5-hydroxytryptophan added to rat adrenal tissue caused a dose-dependent increase in corticosterone production. In a dose of 0.04 microgram/ml, 0.4 microgram/ml and 4.0 microgram/ml, 5-hydroxytryptophan was able to inhibit the ACTH release caused by hypothalamic tissue in vitro. However 0.04 microgram/ml was ineffective on the increase in ACTH secretion elicited by hypothalamic extract CRF. The data suggest that the inhibitory action of 5-hydroxytryptophan and 5-hydroxytryptamine is exerted at the hypothalamic level by inhibiting the release and/or synthesis of the corticotrophic releasing factor.     

Investigation of the corticosteroid receptor system in rat hippocampus by ion exchange fast protein liquid chromatography

In order to study the receptor system for adrenocortical steroids, hippocampal cytosolic preparations--containing both type I and type II receptors--were subjected to anion exchange fast protein liquid chromatography (FPLC). With running buffer containing Tris, EDTA, and glycerol three peaks (1-3) were eluted from the column at 220, 400 and 560 mM NaCl respectively regardless of whether [3H]corticosterone or [3H]RU 28362 had been used as radiotracer. None of the peaks was caused by serum transcortin as revealed by control studies. However, the sequestering influence of transcortin on receptor binding of corticosterone could be demonstrated by the FPLC technique with mixtures containing serum and hippocampus cytosol. Competition experiments with cytosolic samples revealed that type I receptor was present only in peaks 2 and 3 while type II was found in all three peaks in variable amounts, depending on the presence of molybdate. When molybdate was added to the running buffer only two peaks (2 and 3) were eluted, both containing type I and type II receptors. Peak 1 was attributed to the activated type II receptor while peak 2 represented nonactivated receptors. The origin of peak 3 remains uncertain. The data indicate that molybdate must be present in the cytosolic preparation and in the running buffer to keep type II receptor in its nonactivated form. Type I receptor was probably not transformed into the activated form in the absence of molybdate but lost binding capacity and/or affinity for corticosterone.     

The release of corticosterone and a corticosterone-binding protein by incubated rat adrenal slices

Stimulation of incubated rat adrenal slices with ACTH(1-24) resulted in an increase in the release of both corticosterone and specific corticosterone-binding protein into the incubation medium. The release of corticosterone and binding protein was dose and calcium dependent with adrenals from animals pretreated with betamethasone. While the secretion of corticosterone was continuous throughout the incubation period, there appeared to be a limit to the increase in binding capacity. The specificity of steroid binding to the adrenal protein showed a similar profile to that of corticosteroid-binding globulin (CBG) in rat serum. A Western blot analysis using anti-rat CBG as the primary antiserum, showed that the adrenal protein was not CBG. [3H]corticosterone binding with disc electrophoresis, run at 2 degrees C, gave a single peak with approximately the same Rf value for rat serum, purified CBG, and adrenal incubate; at 22 degrees C peaks were only seen for rat serum or purified CBG. The data presented provides further evidence for the existence of a specific corticosterone-binding protein of adrenal origin released in conjunction with corticosterone. The adrenal protein would appear to have a lower affinity for corticosterone than does CBG, and to be functionally more labile. It is possible that the adrenal protein may be CBG that has been internalized, modified and released with corticosterone.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8-10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.     

Development of Adrenocortical Cyclic Nucleotide (Cyclic AMP and Cyclic GMP) and Corticosterone Orcadian Rhythms in Male and Female Rats

The daily rhythm of the adrenocortical cyclic nucleotides (cyclic AMP and cyclic GIMP) was studied in infant male and female Wistar rats before and after the establishment of an adult-like daily rhythm of plasma corticosterone. As in this strain the rhythm of corticosterone is known to be present on postnatal day 18, pups of 2 and 3 weeks of age were studied. The dams and the pups as well as the young adult animals were kept on a controlled 12L-12D photoperiod. Groups of 8–10 pups were killed at 4-hr intervals throughout the day. Plasma corticosterone levels and adrenal cyclic AMP and cyclic GMP concentrations were simultaneously measured and the daily patterns established. Pups of 2 weeks of age showed neither plasma corticosterone nor adrenal cyclic AMP rhythms whereas pups of 3 weeks of age exhibited a typical adult-like circadian rhythm for both variables. The patterns for adrenal cyclic GMP differed according to sex: In female pups no cyclic GMP circadian rhythm could be detected at either 2 or 3 wk. In male pups of 3 wk a typical mature rhythm for adrenal cyclic GMP was evident whereas in younger male pups (2 wk) a circadian rhythm was detected. This circadian rhythm, however, differed from mature circadian rhythm in that its peak was located at 1300 hr instead of 0700 hr. These results demonstrate that, unlike that of cyclic AMP the adrenal cyclic GMP circadian rhythm does not appear at the same time as the plasma corticosterone circadian rhythm. Moreover, a circadian rhythmicity for adrenal cyclic GMP can be found in the absence of any corticosterone circadian rhythm. These facts argue against the view of cyclic GMP being a mediator of ACTH-stimulated steroidogenesis.