Oxytocin in Brattleboro rats: increased synthesis is contrasted by blunted intrahypothalamic release from supraoptic nucleus neurones.

Adult male Brattleboro rats were used to investigate the impact of the congenital absence of vasopressin on the release pattern of oxytocin (OXT) within the hypothalamic supraoptic nucleus (SON) in response to a 10-min forced swimming session and osmotic stimulation. Both immunohistochemical and in situ hybridisation data suggest that vasopressin-deficient animals have more oxytocin-synthesising neurones in the SON than homozygous wild-type controls. Unexpectedly, both forced swimming and peripheral osmotic stimulation resulted in a blunted release profile of oxytocin within the SON of vasopressin-deficient rats compared to controls. A similar intranuclear OXT response to direct osmotic stimulation of the SON by retrodialysis with hypertonic Ringer's solution in both genotypes confirmed the capability of SON neurones to locally release oxytocin in vasopressin-deficient rats, indicating an altered processing of information originating from multisynaptic inputs rather than a deficit in release capacity. Taken together with data obtained in previous studies, the present findings provide evidence suggesting that autocrine and paracrine signalling of magnocellular neurones differs within the paraventricular nucleus and the SON. Thus, significant alterations in intra-SON oxytocin mRNA levels cannot easily be extrapolated to intranuclear release profiles and the local signal intensity of this neuropeptide after physiological stimulation.

Congenital absence of vasopressin and age-dependent changes in ACTH and corticosterone stress responses in rats.

The hypothalamic components of the hypothalamo-pituitary-adrenal axis (HPA) are corticotropin-releasing hormone (CRH) and vasopressin. To test the hypothesis that HPA regulation changes with age, we compared ether and bacterial lipopolysaccharide (LPS) injection induced stress reactions in adult and 10-day-old Brattleboro rats, which naturally lack vasopressin owing to mutation of the gene (di/di). The LPS stimulus was used also with V(1b) receptor antagonist pretreatment (SSR149415). In adult di/di or V(1b) pretreated rats, we observed normal pituitary and adrenocortical secretory responses, while in all 10-day-old rats stress-induced serum corticosterone increases were marked, but adrenocorticotropin (ACTH) increases were significantly smaller. Compared to control pups the adenohypophysis of the 10-day-old di/di rats responded normally to CRH, but their adrenal glands were hyper-responsive to ACTH, while in adults there was greater secretion at both levels with no difference between the genotypes. The serum transcortin level was higher in adults than pups, with the di/di pups having higher transcortin levels than controls. Hence, using the same stressors in adults and pups with both a genetic model and pharmacological pretreatment, we have shown that the role of vasopressin in ACTH regulation is more important during the neonatal period than in adulthood. Blunted hypophysial sensitivity to CRH and similar adrenal gland sensitivity to ACTH in the pups compared to adults suggest that hypothalamic factors could be responsible for the neonatal stress hyporesponsive period.

Tuberoinfundibular peptide of 39 residues- immunoreactive fibers in the zona incerta and the supraoptic decussations terminate in the neuroendocrine hypothalamus.

Tuberoinfundibular peptide of 39 residues (TIP39) is expressed by neurons in the subparafascicular area, the posterior intralaminar complex of the thalamus and the pontine medial paralemniscal nucleus. TIP39-positive fibers from these areas do not form individual bundles or fascicles, they join other pathways to reach their innervated brain areas. Fibers arise from TIP39 perikarya located in the subparafascicular area and the posterior intralaminar complex of the thalamus could be followed to the hypothalamus. After uni- and bilateral posterolateral surgical deafferentations of the hypothalamus, accumulation of TIP39 immunoreactivity was observed in the fibers caudal to the knife cut, while it disappeared completely rostral to the transection. In serial sections of the forebrain, we could follow TIP39-ir fibers coursing within the zona incerta and the supraoptic decussations. TIP39-positive fibers that join the incerto-hypothalamic pathway reach the medio-dorsal part of the hypothalamus and form moderate to high density networks in the dorsomedial and paraventricular nuclei. The other set of TIP39-positive axons from the subthalamic area join the fibers of the supraoptic decussations and run in an antero-medial direction through the most ventral portion of the hypothalamus up to the retrochiasmatic area, where they crossover. A certain portion of these TIP39-positive fibers terminates in the territories of the arcuate and the medial preoptic nuclei, as well as in the retrochiasmatic area.

Signs of attenuated depression-like behavior in vasopressin deficient Brattleboro rats.

Vasopressin, a peptide hormone functioning also as a neurotransmitter, neuromodulator and regulator of the stress response is considered to be one of the factors related to the development and course of depression. In the present study, we have tested the hypothesis that congenital deficit of vasopressin in Brattleboro rats leads to attenuated depression-like behavior in tests modeling different symptoms of depression. In addition, hypothalamic-pituitary-adrenocortical axis activity was investigated. Vasopressin deficient rats showed signs of attenuated depression-like behavior in forced swimming and sucrose preference tests, while their behavior on elevated plus maze was unchanged. Vasopressin deficiency had no influence on basal levels of ACTH and corticosterone and had only mild impact on hormonal activation in response to forced swimming and plus-maze exposure. However, vasopressin deficient animals showed higher level of dexamethasone induced suppression of corticosterone response to restraint stress and higher basal levels of corticotropin-releasing hormone mRNA in the hypothalamic paraventricular nucleus. In conclusion, present data obtained in vasopressin deficient rats show that vasopressin is involved in the development of depression-like behavior, in particular of the coping style and anhedonia. Moreover, behavioral and endocrine responses were found to be dissociated. We suggest that brain vasopressinergic circuits distinct from those regulating the HPA axis are involved in generating depression-like behavior.

Maternal genotype influences stress reactivity of vasopressin-deficient brattleboro rats.

The role of vasopressin, cosecreted with corticotropin-releasing hormone (CRH), in stress is debated, because both normal as well as reduced adrenocorticotropin hormone (ACTH) rise to an acute challenge has been reported in Brattleboro rats genetically lacking vasopressin (di/di). Because di/di pups could be born either from di/+ (heterozygous) or from di/di mothers, and maternal influence is known to modify adult responsiveness, we investigated whether the influence of maternal genotype could explain the variability. Adult rats from mothers with different genotypes were stressed with 60 min restraint and trunk blood was collected for measuring hormone content by radioimmunoassay at the end of stress. All offspring of di/+ mothers had similar ACTH responses to restraint, while the di/di rats born to, and raised by di/di mothers showed reduced ACTH reactivity to restraint. The di/di rats showed elevated water turnover and required a daily cage cleaning every day, which meant frequent handling. To offset the role of handling, all rats had daily cage cleaning in the next series, but the results were the same as in the first series. To investigate whether lactation, the behaviour of the mother or some other factor during the pregnancy is responsible for the differences, pups from di/+ dams were raised by di/di foster mothers and vice versa. We found that the genotype of parental mother is more important than that of the foster mother. The corticosterone and prolactin elevation normally seen after acute stress was unchanged by family history, maternal or personal genotype. Furthermore, in studies with mutant animals, the rearing conditions should be controlled by the experimenter. In experiments with Brattleboro rats, the use of homozygous and heterozygous rats from the same litters of di/+ dams and di/di males is recommended. Our results suggest that vasopressin is not indispensable for ACTH release, and that the di/di genotype of the parental mother can decrease the stress reactivity of the di/di Brattleboro rats.

Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint.

The role of hypothalamic structures in the regulation of chronic stress responses was studied by lesioning the mediobasal hypothalamus or the paraventricular nucleus of hypothalamus (PVH). Rats were acutely (60 min) and/or repeatedly (for 7 days) restrained. In controls, a single restraint elevated the plasma adrenocorticotropin (ACTH), corticosterone, and prolactin levels. Repeated restraint produced all signs of chronic stress, including decreased body and thymus weights, increased adrenal weight, basal corticosterone levels, and proopiomelanocortin (POMC) mRNA expression in the anterior pituitary. Some adaptation to repeated restraint of the ACTH response, but not of other hormonal responses, was seen. Lesioning of the mediobasal hypothalamus abolished the hormonal response and POMC mRNA activation to acute and/or repeated restraint, suggesting that the hypothalamo-pituitary-adrenal axis activation during repeated restraint is centrally driven. PVH lesion inhibited the ACTH and corticosterone rise to the first restraint by approximately 50%. In repeatedly restrained rats with PVH lesion, the ACTH response to the last restraint was reduced almost to basal control levels, and the elevation of POMC mRNA level was prevented. PVH seems to be important for the repeated restraint-induced ACTH and POMC mRNA stimulation, but it appears to partially mediate other restraint-induced hormonal changes.

Rhodopsin-transducin interface: studies with conformationally constrained peptides.

To probe the interaction between transducin (G(t)) and photoactivated rhodopsin (R*), 14 analog peptides were designed and synthesized restricting the backbone of the R*-bound structure of the C-terminal 11 residues of G(t)alpha derived by transferred nuclear Overhauser effect (TrNOE) NMR. Most of the analogs were able to bind R*, supporting the TrNOE structure. Improved affinities of constrained peptides indicated that preorganization of the bound conformation is beneficial. Cys347 was found to be a recognition site; particularly, the free sulfhydryl of the side chain seems to be critical for R* binding. Leu349 was another invariable residue. Both Ile and tert-leucine (Tle) mutations for Leu349 significantly reduced the activity, indicating that the Leu side chain is in intimate contact with R*. The structure of R* was computer generated by moving helix 6 from its position in the crystal structure of ground-state rhodopsin (R) based on various experimental data. Seven feasible complexes were found when docking the TrNOE structure with R* and none with R. The analog peptides were modeled into the complexes, and their binding affinities were calculated. The predicted affinities were compared with the measured affinities to evaluate the modeled structures. Three models of the R*/G(t)alpha complex showed strong correlation to the experimental data.

Various ulcerogenic stimuli are potentiated by glucocorticoid deficiency in rats.

The effects of glucocorticoid deficiency with or without corticosterone replacement on susceptibility to gastric mucosal injury by various ulcerogenic stimuli have been evaluated in rats. Gastric erosions were induced in male rats by stimuli of different modalities and intensities: 20% ethanol (po), aspirin (300 mg/kg, ip), acidified aspirin (40 mM, po) and 100% acetic acid (applied to gastric serosa). Glucocorticoid supply was decreased by adrenalectomy or by delayed inhibitory action after a single pharmacological dose of cortisol (300 mg/kg, ip) injected one week before the onset of ulcerogenic stimulus. Corticosterone for replacement (4 mg/kg, sc) was injected in rats with glucocorticoid deficiency 15 min before the onset of ulcerogenic stimulus. Plasma corticosterone levels were measured by fluorometry. Gastric erosions were quantitated by measuring the area of damage. Ulcerogenic stimuli induced both plasma corticosterone rise and gastric mucosal injury. The area of mucosal damages induced various stimuli ranged from small to extensive. Glucocorticoid deficiency significantly potentiated an ulcerogenic action of every ulcerogenic stimulus. Replacing corticosterone prevented or significantly decreased erosion-potentiating effect of glucocorticoid deficiency. These results show that endogenous glucocorticoids released during ulcerogenic influences help gastric mucosa to resist a harmful action of both weak and strong ulcerogenic stimuli.

Gastroprotective action of glucocorticoids during the formation and the healing of indomethacin-induced gastric erosions in rats.

The aim of the present study consisted of the investigation of glucocorticoid role in the formation and the healing of indomethacin-induced (25 mg/kg, s.c.) gastric erosions in rats. The effect of deficiency of glucocorticoid production followed by corticosterone replacement on the formation and the healing of the gastric erosions was evaluated. Glucocorticoid production was decreased by adrenalectomy or by delayed inhibitory action after a single pharmacological dose of cortisol (300 mg/kg i.p.) injected 1 week before the onset of ulcerogenic stimulus. Indomethacin induced corticosterone rise and caused gastric erosions. The loss of indomethacin-induced plasma corticosterone rise potentiated the formation of indomethacin-induced erosions in both models. The area of gastric erosions in rats with glucocorticoid deficiency was considerably larger than that in control animals 4 h after indomethacin administration as well as during 48 h after the drug administration (period of erosion healing). Injecting corticosterone in rats with glucocorticoid deficiency significantly decreased the formation of indomethacin-induced gastric erosions and promoted their healing. Thus, the present data support the gastroprotective action of glucocorticoids in the formation and in the healing of indomethacin-induced mucosal injury.

The effect of glucocorticoids on the anxiolytic efficacy of buspirone.

RATIONALE: The serotonergic system and the hypothalamus-hypophysis-adrenocortical axis reciprocally influence each other. Therefore, the interaction between stress and serotonergic anxiolytics should be of major concern for both laboratory investigations and clinical treatment. OBJECTIVES: We have studied the effects of the serotonergic anxiolytic buspirone in rats in which basal levels of glucocorticoids were low and stable, while acute stress reactions were inhibited or exogenously induced. METHODS: Rats were adrenalectomised. Subcutaneous corticosterone pellets maintained basal glucocorticoid concentrations while acute changes were mimicked by corticosterone injections. Anxiety was assessed by the social interaction test. Temporal changes were evaluated by submitting rats to the same manipulations three times at two-day intervals. RESULTS: Buspirone applied to animals with stable and low plasma glucocorticoid concentrations induced a dramatic increase in social interactions. A slight locomotor suppressive effect was also noticed. The effects of buspirone proved to be stable over time in these animals. Acute treatment with corticosterone doubled the locomotor suppressive effects of buspirone and reversed its anxiolytic effects: the buspirone-corticosterone combination was anxiogenic after the first application. During the second and third treatment, the impact of corticosterone on buspirone efficacy gradually decreased, but the combined treatment remained about half as effective in reducing anxiety as buspirone alone.

Measuring molecular similarity and diversity: total pharmacophore diversity.

A novel method, total pharmacophore diversity (ToPD), based on known pharmacophore features for numerically defining molecular similarity or diversity is described. The method captures the 3D shape and functionality of molecules by the analysis of relevant intramolecular distances to generate a short and descriptive pharmacophoric fingerprint for each molecule. The ToPD fingerprints can then be used in diversity analysis, clustering, or database searching. Conformational sampling is carried out when needed by the means of molecular dynamics. Our results show that ToPD outperforms a traditional 2D fingerprint technique in all test cases.

Synthesis of bicyclic pyrimidine derivatives as ATP analogues.

A highly efficient and general solid-phase synthesis of bicyclic pyrimidine derivatives that target purine dependent proteins is reported. The synthesis of the key intermediate, 4,6-disubstituted-5-amino-pyrimidine, involved reduction of the corresponding nitro derivatives using 1,1'-dioctyl-viologen in a triphasic milieu. The mild reduction conditions enable the use of any acid labile solid support as well as a wide range of combinatorial substituents, thus enabling the synthesis of large libraries of highly diverse bicyclic pyrimidines. Alternative reduction conditions with tin(II) chloride and structure-reactivity studies are discussed as well.

Non-genomic effects of glucocorticoids in the neural system. Evidence, mechanisms and implications.

Complementing the classical concept of genomic steroid actions, here we (i) review evidence showing that important neural effects of glucocorticoids are exerted by non-genomic mechanisms; (ii) describe known mechanisms that may underlie such effects; (iii) summarize the functions and implications of non-genomic mechanisms and (iv) outline future directions of research. The role of non-genomic mechanisms is to shape the response of the organism to challenges that require a substantial reorganization of neural and somatic functions and involve massive behavioral shifts. Non-genomic effects may (i) prepare the cell for subsequent glucocorticoid-induced genomic changes, (ii) bridge the gap between the early need of change and the delay in the expression of genomic effects and (iii) may induce specific changes that in some instances are opposite to those induced by genomic mechanisms. The latter can be explained by the fact that challenging situations require different responses in early (acute) and later (chronic) phases. Data show that non-genomic mechanisms of glucocorticoid action play a role in both pathological phenomena and the expression of ameliorative pharmacological effects. Non-genomic mechanisms that underlie many glucocorticoid-induced neural changes constitute a for long overlooked controlling factor. Despite the multitude and the variety of accumulated data, important questions remain to be answered.

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.

The effect of hypothalamic lesions on hypothalamo-pituitary-adrenal axis activity and inflammation in adjuvant-induced arthritis.

Adjuvant-induced arthritis (AA) was induced in control and in hypothalamic lesioned Piebald-Viral-Glaxo (PVG) rats. Following discrete paraventricular nucleus (PVN) lesions plasma corticosterone was increased 14 days after adjuvant injection as in controls, when hind paw inflammation was apparent. PVN lesion did not affect the severity of inflammation.In contrast, following medial basal hypothalamus (MBH) lesions adjuvant did not increase corticosterone levels and the increase in paw volume at day 14 was potentiated. Basal proopiomelanocortin(POMC) mRNA expression in the anterior lobe was unchanged by PVN lesions and decreased by MBH lesions. AA increased POMC mRNA in controls and in both PVN and MBH lesioned rats. After complete MBH lesion, surviving anterior pituitary tissue maintained morning levels of corticosterone.Thus, AA may activate the hypothalamo-pituitary-adrenal axis without the mediation of PVN neurones projecting to the median eminence. However, the loss of the corticosterone response to AA and the increase in severity of inflammation in the MBH lesioned rats suggests a central (non-PVN) component mediates effects of inflammation. Furthermore, the increase in POMC mRNA in the MBH lesioned AA rats suggests that part of this process is not mediated by releasing factors in the hypothalamo-hypophysial portal system, and that extrahypothalamic(peripheral) mediators act on the pituitary during chronic inflammation.

Housing conditions and the anxiolytic efficacy of buspirone: the relationship between main and side effects.

Serotonergic anxiolytics yield contradictory results both in the laboratory and clinically. In an attempt to investigate the cause of discrepancies, the anxiolytic effect of buspirone (0, 3 or 10 mg/kg, single treatment) was tested 1 h and 4 h after injection in rats in different housing conditions. At 1 h after drug administration, buspirone increased corticosterone production and decreased locomotor behaviour in both the elevated plus-maze and the social interaction tests. No anxiolytic-like effect was produced in either test. At 4 h after drug injection, no corticosterone or locomotor effects of buspirone were observed. In contrast, anxiolytic effects emerged in this phase. Open arm exploration and social investigation were increased in the plus-maze and social interaction test, respectively. In the plus-maze, the anxiolytic effect was significant in isolated animals only. In the social interaction test, the anxiolytic effect was stronger in isolated than in group-housed animals. When corticosterone secretion was inhibited by adrenalectomy, a full anxiolytic effect of buspirone was observed 1 h after drug administration. It appears that the side effects of buspirone have a shorter duration than the main anxiolytic effect. The buspirone-induced increase in corticosterone may have abolished the anxiolytic effects of the drug shortly after injection. Individual housing enhanced the anxiolytic efficacy of buspirone 4 h after administration.

Radiodetoxified lipopolysaccharide fails to activate the hypophyseal- pituitary-adrenal axis in the rat.

Lipopolysaccharide (LPS) is known to raise the concentration of the circulating stress hormones such as ACTH, corticosterone and beta-endorphin. This effect of endotoxin is mediated by different immune system-released hormone-like factors (e.g. interleukins, tumor necrosis factor etc.). Gamma-ray irradiation of LPS alters its biological properties and results in a radiodetoxified LPS preparation with numerous beneficial effects and decreased toxicity. In this study we compared the neuroendocrine effects of a commercial LPS and our native and radiodetoxified LPS preparations in rats. Plasma ACTH, corticosterone and beta-endorphin levels were measured by specific radioimmunoassays 120 min after intraperitoneal LPS administration. Control animals were injected with saline. Results show a dramatic increase in all hormones after administration of commercial and our native LPS preparation. Hormone levels in saline-injected controls and radiodetoxified LPS-treated rats did not rise significantly. These results suggest that radio-detoxification disintegrated that part of the LPS molecule complex which is responsible for toxicity including an enhanced production of cytokines, which trigger the hypothalamo-pituitary-adrenal axis.

Ultradian corticosterone rhythm and the propensity to behave aggressively in male rats.

Ultradian fluctuations in plasma glucocorticoids have been demonstrated in a variety of species including humans. The significance of such rhythms is poorly known, although disorganized ultradian glucocorticoid rhythms have been associated with behavioural disorders. Here we report that ultradian glucocorticoid rhythms may establish the propensity to behave aggressively in male rats. Male rats were significantly more aggressive in the increasing phase of their corticosterone fluctuation when confronting a male intruder than counterparts in the decreasing phase of their corticosterone fluctuations facing such opponents. Corticosterone fluctuations were mimicked by a combination of treatments with the corticosterone synthesis inhibitor metyrapone and corticosterone. Again, males with increased plasma corticosterone levels were more aggressive than counterparts with a decreased plasma corticosterone concentration. These data suggest that the behavioural response to an aggressive challenge may vary in the same animal across the day due to the pulsating nature of corticosterone secretion. Aggressive behaviour is also episodic in humans; moreover, intermittent explosive behaviour is recognized as a psychological disorder. It can be hypothesized that a temporal coincidence between the occurrence of a challenge and a surge in plasma corticosterone concentration may be one of the factors that promote episodic aggressive outbursts.

Defeat is a major stressor in males while social instability is stressful mainly in females: towards the development of a social stress model in female rats.

Social stress models appear useful in elucidating the interrelationship between stress, mood disorders, and drug efficacy. However, reliable social stress models for females are virtually lacking. The aim of this study was to determine stress-related consequences of (a) defeat in aggressive encounters and (b) social instability, in male and female rats. Defeat in male and female subjects was induced by aggressive male residents and female residents made aggressive by surgery (mediobasal hypothalamic lesion [MBHL]), respectively. Aggressiveness of resident males and resident MBHL females was remarkably similar. Alternating isolation and mixed-sex crowding phases with membership rotation were used to induce social instability. Aggression was kept low in the latter paradigm by manipulating crowding group composition. Defeat stress reduced weight gain, and increased both adrenals and plasma corticosterone in males. Only adrenal weight was affected in females. Social instability reduced weight gain, and induced thymus involution, adrenal hypertrophy and elevated plasma corticosterone levels in females. Only weight gain and thymus weights were affected in males. It is concluded that defeat stresses males more than females, while social instability is more stressful for females than for males, if aggressive contacts are low. It is suggested that the social instability model is a good model of social stress in females.

Effect of cholinergic drugs on the concentration of intracellular free calcium of rat pituitary intermediate lobe cells.

We tested the effect of cholinergic drugs on the concentration of intracellular free calcium in rat melanotropes. Acetylcholine, muscarine, carbachol, and nicotine resulted in a significant rise in this parameter. Effect of acetylcholine was reduced by atropine (non-selective muscarinic antagonist), pirenzepine (M1 muscarinic antagonist), and 4-DAMP (M3 > M1 muscarinic antagonist), but exposure to the M1 muscarinic agonist McN-A 343 resulted in a significantly smaller calcium-response than that seen in response to acetylcholine or to muscarine. This suggests the involvement of both M1 and M3 muscarinic receptors in the acetylcholine-induced calcium-rise. On the other hand, in the presence of atropine the acetylcholine-induced calcium-rise was not eliminated: this fact indicates that nicotinic receptors are also involved in the acetylcholine-induced intracellular calcium-rise. The acetylcholine-, and nicotine-induced calcium-rise was significantly reduced in presence of the neuronal-type nicotinic antagonist, mecamylamine. This suggests the involvement of a neuronal-type nicotinic receptor in the acetylcholine-induced intracellular calcium-response. Moreover, because in a further experiment almost 80% of the cells investigated responded to muscarine as well as nicotine, we conclude that both functionally active muscarinic and nicotinic receptors are present on the same cell.