Chronic treatment with enhancer drugs modifies the gene expression of selected parameters related to brain plasticity in rats under stress conditions.

Selegiline, also known as L-deprenyl, and (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP) were found to induce enhancement of monoamine neurotransmission in low and very low doses. In addition, these enhancers may modify glutamatergic neurotransmission. The aim of the present study was to test the hypothesis that under stress conditions, chronic treatment with enhancer drugs has a positive impact on the glutamatergic system and other parameters related to brain plasticity, stress-related systems, and anxiety behavior. We exposed male Wistar rats to a chronic mild stress procedure combined with chronic treatment with two synthetic enhancer drugs. The gene expression of GluR1, an AMPA receptor subunit was reduced by repeated treatment with deprenyl in the hippocampus and with both BPAP and deprenyl in the prefrontal cortex. A significant reduction of NMDA receptor subunit GluN2B expression was observed in the hippocampus but not in the prefrontal cortex. Deprenyl treatment led to an enhancement of hippocampal BDNFmRNA concentrations in stress-exposed rats. Treatment with enhancer drugs failed to induce significant changes in stress hormone concentrations or anxiety behavior. In conclusion, the present study in chronically stressed rats showed that concomitant treatment with enhancer drugs did not provoke substantial neuroendocrine changes, but modified gene expression of selected parameters associated with brain plasticity. Observed changes may indicate a positive influence of enhancer drugs on brain plasticity, which is important for preventing negative consequences of chronic stress and enhancement of stress resilience. It may be suggested that the changes in glutamate receptor subunits induced by enhancer drugs are brain region-specific and not dose-related.

The prepulse inhibition deficit appearance is largely independent on the circadian cycle, body weight, and the gender of vasopressin deficient Brattleboro rat.

OBJECTIVE: A disturbance of sensorimotor gating measured by prepulse inhibition of acoustic startle (PPI) is one of the best tests of the schizophrenia-like behavior. Vasopressin was implicated in the development of schizophrenia; therefore, the naturally occurring vasopressin-deficient Brattleboro rat has been suggested to be a reliable non-pharmacological animal model. However, previous studies focusing on PPI deficit did not use proper control and despite clear gender differences in the development of the disorder, the effect of gender has been mostly neglected. METHODS: First, we compared the "noise" and "tone" type prepulse at 73-77-81 dB intensity during the light or dark phase using small (~150 g) or big (~500 g) Wistar rats. The test parameters were validated by a pharmacological schizophrenia model (30 mg/kg ketamine i.p.). Than male, female, and lactating vasopressin-deficient animals were compared with +/+ ones. RESULTS: We established that the prepulse "noise" type is not optimal for PPI testing. The cycle of the day as well as the body weight had no effect on PPI. Even if we compared vasopressin-deficient animals with their closely related +/+ controls, the PPI deficiency was visible with more pronounced effect at 77 dB prepulse intensity similarly to pharmacological schizophrenia model. Despite our expectation, the gender as well as lactation had no effect on the vasopressin-deficiency induced PPI deficit. CONCLUSIONS: The present data confirmed and extended our previous studies that vasopressin-deficient rat is a good model of schizophrenia. It seems that female as well as lactating Brattleboro rats are useful tools for testing putative novel antipsychotics in line with special attention required for schizophrenic women.

A wider view on gastric erosion: detailed evaluation of complex somatic and behavioral changes in rats treated with indomethacin at gastric ulcerogenic dose.

OBJECTIVE: Gastric erosion is widespread side effect of nonsteroidal anti-inflammatory drugs. To examine the complexity of the brain-gut axis regulation, indomethacin-induced gastric erosion formation was studied in connection with somatic and behavioral changes. METHODS: During a constant telemetric recording of heart rate, body temperature, and locomotion of male rats we examined the effects of 24 h fasting, indomethacin (35 mg/kg s.c.) injection, and refeeding at 4 h. Behavior was analyzed on elevated plus maze (EPM) at 24 h and somatic changes at 72 h. RESULTS: Gastric erosion developed 4 h after indomethacin injection, healed 72 h later contrasted by large injury in the small intestine. As classical signs of chronic stress, body and thymus weight were reduced while adrenal weight was enhanced 72 h after indomethacin injection. Fasting by itself changed all telemetrically recorded parameters with most prominent decrease in heart rate. Indomethacin induced similar diminishing effects with earliest and strongest temperature decrease. As a sign of more anxious phenotype locomotion reducing effect of indomethacin injection was detected on EPM. The EPM-induced temperature elevation was missing in indomethacin-treated animals. CONCLUSIONS: Fasting by itself induce somatic changes, which can make the animals more vulnerable to ulcerogenic stimuli. Development of indomethacin-induced gastrointestinal lesions happened in parallel with disturbances of heart rate, core body temperature, and chronic stress-like somatic changes as well as anxiety-like behavior. We have to be more aware of the existence of the brain-gut axis and should study changes in the whole body rather than focusing on a specific organ. elevated plus maze.

Early life adversity and serotonin transporter gene variation interact at the level of the adrenal gland to affect the adult hypothalamo-pituitary-adrenal axis.

The short allelic variant of the serotonin transporter (5-HTT) promoter-linked polymorphic region (5-HTTLPR) has been associated with the etiology of major depression by interaction with early life stress (ELS). Furthermore, 5-HTTLPR has been associated with abnormal functioning of the stress-responsive hypothalamo-pituitary-adrenal (HPA) axis. Here, we examined if, and at what level, the HPA-axis is affected in an animal model for ELS × 5-HTTLPR interactions. Heterozygous and homozygous 5-HTT knockout rats and their wild-type littermates were exposed daily at postnatal days 2-14 to 3 h of maternal separation. When grown to adulthood, plasma levels of adrenocorticotropic hormone (ACTH), and the major rat glucocorticoid, corticosterone (CORT), were measured. Furthermore, the gene expression of key HPA-axis players at the level of the hypothalamus, pituitary and adrenal glands was assessed. No 5-HTT genotype × ELS interaction effects on gene expression were observed at the level of the hypothalamus or pituitary. However, we found significant 5-HTT genotype × ELS interaction effects for plasma CORT levels and adrenal mRNA levels of the ACTH receptor, such that 5-HTT deficiency was associated under control conditions with increased, but after ELS with decreased basal HPA-axis activity. With the use of an in vitro adrenal assay, naïve 5-HTT knockout rats were furthermore shown to display increased adrenal ACTH sensitivity. Therefore, we conclude that basal HPA-axis activity is affected by the interaction of 5-HTT genotype and ELS, and is programmed, within the axis itself, predominantly at the level of the adrenal gland. This study therefore emphasizes the importance of the adrenal gland for HPA-related psychiatric disorders.

Does chronic stress enhance the risk of diseases?

OBJECTIVE: In the everyday life, stress is deemed as something unfavorable that may enhance the risk for the development or worsen a disease. However, in its nature, stress is adaptive reaction of the body. Its main characteristic is the activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Previously, we have shown that activation of the HPA axis plays a gastroprotective role during acute stress. The aim of our study was to clarify the effects of chronic stress and chronically elevated basal corticosterone levels on the gastric ulceration and cardiovascular vulnerability in rats. METHODS: Male Wistar rats were repeatedly restrained 60 min daily for 14 days and examined on day 15th. The gastric ulceration was induced by a s.c. injection of indomethacin (35 mg/kg). The cardiovascular vulnerability was examined in urethane-anaesthetized rats in an experimental angina pectoris model (epinephrine, 10 µg/kg, 30 s later phentolamine, 15 mg/kg, both i.v.). RESULTS: We confirmed the development of chronic stress consequences by changes in several somatic parameters (body weight decrease, thymus involution, adrenal gland hypertrophy), and elevated resting corticosterone levels. However, the gastroprotective effect of chronic stress was not manifested and there was no aggravation of indomethacin-induced gastric ulceration, either. In the experimental angina pectoris model, previous chronic stress did not have any profound effect on the blood pressure, heart rate, and electrocardiogram changes. CONCLUSIONS: In contrast to the general view on the harmfulness nature of the stress, we were unable to find a harmful effect of chronic stress on the internal diseases (gastric ulceration and angina pectoris). However, its protective effect was also missing among present experimental conditions.

Role of ionotropic glutamate receptors in the control of prolactin secretion by other neurotransmitters and neuropeptides at the level of the pituitary.

OBJECTIVE: Although prolactin (PRL) plays an important role in the milk production, it has also many other functions. PRL secretion can be inhibited by dopamine and stimulated by serotonin, thyrotropin releasing hormone (TRH), and vasoactive intestinal peptide (VIP). However, the exact mechanisms of PRL regulation are still not fully understood. Glutamate is also a potent elevator of PRL secretion. It has several receptors: ionotropic NMDA (N-methyl-D-aspartate) and non-NMDA as well as metabotropic receptors. Our interest was to find out whether endogenous glutamate may act at the hypophyseal level and affect the PRL regulating neurotransmitters (dopamine, serotonin, TRH, VIP). METHODS: MK-801 as NMDA blocker and GYKI52466 as a non-NMDA antagonist were used in this study. For dopamine and serotonin experiments intraperitoneal drug administration and blood sampling were applied. On the other hand, TRH and VIP effect on PRL secretion was studied in in vitro conditions by incubating them with a half 300*300nm choppered anterior pituitary gland of the adult male rats. RESULTS: The basal PRL levels were not influenced by the glutamate antagonists used either alone or combined in both in vivo and in vitro conditions. We failed to reveal interaction between dopamine-, serotonin-, and TRH-induced PRL rise and antagonists treatment. MK-801 had a significant inhibitory effect on VIP-induced PRL changes. CONCLUSIONS: Our data confirmed the regulatory role of dopamine, serotonin, and TRH on PRL secretion, however, the interaction between these and glutamatergic systems was not confirmed, at least not via the ionotropic receptors. On the other hand, the endogenous glutamate can through the NMDA receptor subtype contribute to the VIP-induced PRL secretion at the level of the anterior pituitary. This regulation may be especially important during suckling and stress response when rapid release of PRL is required.

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.

Cannabinoid modulation of midbrain urocortin 1 neurones during acute and chronic stress.

Neurones in the centrally projecting Edinger-Westphal nucleus (EWcp) are the main site of urocortin 1 (Ucn1) synthesis in the mammalian brain, and are assumed to play a role in the stress response of the animal. Because endocannabinoid signalling has also been strongly implicated in stress, we hypothesised that endocannabinoids may modulate the functioning of the urocortinergic EWcp. First, using in situ hybridisation, we demonstrated cannabinoid receptor 1 (CB1R) mRNA expression in mouse EWcp-neurones that were Ucn1-negative. Dual- and triple-label immunocytochemistry revealed the presence of CB1R in several GABA-immunopositive fibres juxtaposed to EWcp-Ucn1 neurones. To test functional aspects of such an anatomical constellation, we compared acute (1 h of restraint) and chronic (14 days of chronic mild stress) stress-induced changes in wild-type (WT) and CB1R knockout (CB1R-KO) mice. Acute and especially chronic stress resulted in an increase in Ucn1 content of the EWcp, which was attenuated in CB1R-KO mice. CB1R-KO mice had higher basal and chronic stress-induced adrenocorticotrophin and corticosterone levels and were more anxious on the elevated plus-maze versus WT. Collectively, our results show for the first time EWcp-Ucn1 neurones are putatively innervated by endocannabinoid sensitive, inhibitory, GABAergic afferents. In addition, we provide novel evidence that the absence of the CB1 receptor alters the Ucn1 mRNA and peptide levels in EWcp neurones, concomitant with an augmented stress response and increased anxiety-like behaviour.

The biochemical complexity of the endocannabinoid system with some remarks on stress and related disorders: a minireview.

Nowadays, the endocannabinoid-regulated processes are in the focus of interest, among others, for the treatment of stress-related disorders. In this minireview, we attempt to give some possible explanations for the conflicting results of the cannabinoidergic regulation of the hypothalamo-pituitary-adrenocortical (HPA) axis and related disorders, drawing attention to the complexity of the endocannabinoid system. The endocannabinoid system is a part of an intricate network of lipid pathways and consists of the cannabinoid receptors, their endogenous ligands, and the enzymes catalyzing their formation and degradation. The stress research is focused almost exclusively on the anandamide and 2-arachidonyl glycerol, and the cannabinoid 1 receptor. However, physiological, pathological, and pharmacological perturbations of the interconnected lipid pathways have a profound effect on the regulation of the endocannabinoid signaling system. For example, diet may substantially influence the lipid composition of the body. Recent studies have indicated that beside cannabinoid 1 receptor, the endocannabinoids may act on the cannabinoid 2, peroxisome proliferator-activated, and transient receptor potential of vanilloid type-1 receptors, too. All of these receptors are implicated in the development of stress-related disorders. However, it has to be mentioned that degradation of the endocannabinoids may result in the production of active compounds as well. Since endocannabinoids have a widespread distribution in the body, they may influence a phenomenon at several points. Different effects (stimulatory or inhibitory) at different levels of endocannabinoids (e.g. hypothalamus, hypophysis, adrenal gland in the case of HPA axis) may explain some of their unequivocal results.

Nesfatin-1 exerts long-term effect on food intake and body temperature.

OBJECTIVE: To determine whether the anorexigenic peptide, nesfatin-1 affects energy expenditure, and to follow the time course of its effects. DESIGN: Food intake duration, core body temperature, locomotor activity and heart rate of rats were measured by telemetry for 48 h after a single intracerebroventricular injection of 25 or 100 pmol nesfatin-1 applied in the dark or the light phase of the day. Body weight, food and water intake changes were measured daily. Furthermore, cold-responsive nesfatin-1/NUCB2 neurons were mapped in the brain. RESULTS: Nesfatin-1 reduced duration of nocturnal food intake for 2 days independently of circadian time injected, and raised body temperature immediately, or with little delay depending on the dose and circadian time applied. The body temperature remained higher during the next light phases of the 48 h observation period, and the circadian curve of temperature flattened. After light phase application, the heart rate was elevated transiently. Locomotion did not change. Daily food and water intake, as well as body weight measurements point to a potential decrease in all parameters on the first day and some degree of compensation on the second day. Cold-activated (Fos positive) nesfatin-1/NUCB2 neurones have been revealed in several brain nuclei involved in cold adaptation. Nesfatin-1 co-localised with prepro-thyrotropin-releasing hormone in cold responsive neurones of the hypothalamic paraventricular nucleus, and in neurones of the nucleus raphe pallidus and obscurus that are premotor neurones regulating brown adipose tissue thermogenesis and skin blood flow. CONCLUSION: Nesfatin-1 has a remarkably prolonged effect on food intake and body temperature. Time course of nesfatin-1's effects may be varied depending on the time applied. Many of the nesfatin-1/NUCB2 neurones are cold sensitive, and are positioned in key centres of thermoregulation. Nesfatin-1 regulates energy expenditure a far more potent way than it was recognised before making it a preferable candidate anti-obesity drug.

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.

Gender specific influence of endogenous glutamate release on stress-induced fear in rats.

OBJECTIVE: Stress, fear and anxiety are among major public health concerns. The role of glutamate in these processes is becoming more recognized with promising new drug targets. The aim of this study was to establish the gender specificity of a possible treatment of fear by glutamate antagonists in correspondence with changes in stress-hormone release. METHODS: Footshock-induced fear was used as an anxiogenic situation in rats. A combination of two ionotrop receptor antagonists such as MK-801 (dizocilpine; 0.2 mg/kg) for NMDA (N-methyl-D-aspartic acid) and GYKI 52466 (benzodiazepine derivative; 10 mg/kg) for AMPA/kainate receptors were used for 5 days following the hypothesis that they potentiate each other the main action, but at the same time the side effects may be minimized. RESULTS: Female rats tried to avoid the electrical stimulus more actively than males, as they spent more time with exploration and jumping and less time with freezing or rest. Ionotropic glutamate receptor antagonists have anxiolytic action. MK-801 was more effective in females, as it prevented the footshock-induced freezing per se, while in males it was effective only in combination with GyKI 52466. The locomotor side effect of MK-801 was not visible after repeated administration. The freezing behavior was positively correlated with the changes in prolactin but not with adrenocorticotropin levels. CONCLUSIONS: We proved the involvement of endogenous glutamate neurotransmission in stress-induced fear. Therapeutical usage may involve a combination of different receptor antagonists. Special attention should be paid to the gender, as females seem to be more sensitive, therefore they require smaller doses. During the treatment the prolactin levels should be monitored.

The healing of NSAID-induced gastric lesion may be followed by small intestinal and cardiovascular side effects.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among some of the most commonly used medications. Serious adverse effects induced by NSAIDs may occur not only in the upper gastrointestinal tract, but in the small intestine and cardiovascular system. However, these side effects are studied and investigated separately. Here we tested the hypothesis that the healing of indomethacin-induced gastric erosion may be followed by small intestinal and cardiovascular adverse effects. First we examined the development of gastrointestinal lesions 4-24-48-72 h after a single indomethacin (35 mg/kg s.c.) injection given to fasted male rats (refeeding after 4 h). Then with a telemetric device heart rate, core body temperature and locomotion changes were recorded in the freely moving animals for 72 h after indomethacin or its vehicle injection (control). Indomethacin produced hemorrhagic erosion in the glandular stomach 4 h after its administration which was almost completely healed 48 h later. Parallel to the healing a gradual increase of injury to the small intestine became apparent. The control rats' heart rate, core body temperature and locomotion all agreed with a normal circadian rhythm. However, the circadian cycle of rats treated with indomethacin in 24 h after its administration was disrupted: their heart rate rose to it's maximal level and their locomotion and core temperature values fell to their minimal. These results suggest that the healing of gastric erosion induced by a single indomethacin injection may be followed by other pathological events outside of the stomach, among which there may be intestinal injury and a loss of a normal circadian cycle of heart rate as well as body temperature and locomotion.

The α2-adrenoceptors do not modify the activity of tyrosine hydroxylase, corticoliberine, and neuropeptide Y producing hypothalamic magnocellular neurons ion the Long Evans and Brattleboro rats.

The hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei are activated by body salt-fluid variations. Stimulation of alpha(2)-adrenoceptors by an agonist-xylazine (XYL) activates oxytocinergic but not vasopressinergic magnocellular neurons. In this study, tyrosine hydroxylase (TH), corticoliberine (CRH), and neuropeptide Y(NPY) magnocellular phenotypes, were analysed in response to alpha(2)-adrenoceptor manipulations and sustained hyperosmolality in vasopressin deficient homozygous Brattleboro (di/di) rats. Saline (0.9% NaCl, 0.1 ml/100g/bw), XYL (10 mg/kg/bw), atipamezole (ATIP, alpha(2)-adrenoceptors antagonist, 1 mg/kg/bw), and ATIP 5 min later followed by XYL, were applied intraperitoneally. Presence of immunolabeled Fos peptide signalized the neuronal activity. Ninety minutes after injections, the rats were anesthesized and sacrificed by transcardial perfusion with fixative. Coronal sections of 30 mum thickness double immunolabeled with Fos/neuropeptide were evaluated under light microscope. Under basal conditions, di/di in comparison with control Long Evans rats, displayed significantly higher number of TH, CRH, and NPY immunoreactive neurons in the SON and PVN (except NPY cells in PVN) and more than 90%, 75%, and 86% of TH, NPY, and CRH neurons, respectively, displayed also Fos signal in the SON. XYL did not further increase the number of Fos in the PVN and SON and ATIP failed to reduce the stimulatory effect of hypertonic saline in all neuronal phenotypes studied. Our data indicate that hyperosmotic conditions significantly influence the activity of TH, CRH, and NPY magnocellular neuronal phenotypes, but alpha(2)-adrenoceptors do not play substantial role in their regulation during osmotic challenge induced by AVP deficiency.

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.

The role of endogenous cannabinoids in the hypothalamo-pituitary-adrenal axis regulation: in vivo and in vitro studies in CB1 receptor knockout mice.

Exogenous cannabinoids affect multiple hormonal systems including the hypothalamo-pituitary-adrenocortical (HPA) axis. These data suggest that endogenous cannabinoids are also involved in the HPA control; however, the mechanisms underlying this control are poorly understood. We assessed the role of endogenous cannabinoids in the regulation of the HPA-axis by studying CB1 receptor knockout (KO) and wild type (WT) mice. Basal and novelty stress-induced plasma levels of adrenocorticotropin (ACTH) and corticosterone were higher in CB1-KO than in WT mice. We investigated the involvement of the pituitary in the hormonal effects of CB1 gene disruption by studying the in vitro release of ACTH from anterior pituitary fragments using a perifusion system. Both the basal and corticotropin releasing hormone (CRH)-induced ACTH secretion were similar in CB1-KO and WT mice. The synthetic glucocorticoid, dexamethasone suppressed the CRH-induced ACTH secretion in both genotypes; thus, the negative feedback of ACTH secretion was not affected by CB1 gene disruption. The cannabinoid agonist, WIN 55,212-2 had no effects on basal and CRH-stimulated ACTH secretion by anterior pituitary slices. In our hands, the disruption of the CB1 gene lead to HPA axis hyperactivity, but the pituitary seems not to be involved in this effect. Our data are consistent with the assumption that endogenous cannabinoids inhibit the HPA-axis via centrally located CB1 receptors, however the understanding of the exact underlying mechanism needs further investigation.

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.

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.

Simultaneous blockade of two glutamate receptor subtypes (NMDA and AMPA) results in stressor-specific inhibition of prolactin and corticotropin release.

Many neurons express simultaneously two or more isotypes of glutamate receptors, so that pharmacological modulation of more than one receptor may be necessary to reveal the role of glutamate in mediating physiological processes. The present studies were aimed at evaluating involvement of endogenous glutamate in triggering plasma prolactin (PRL) and adrenocorticotropic hormone (ACTH) levels in response to three different stress stimuli (footshock, immobilization and ether stress). Blockade of glutamate receptor subtypes was achieved by the administration of the NMDA antagonist dizocilpine (MK-801, 0.2 mg/kg) and the selective AMPA antagonist GYKI 52466 (10 mg/kg). Rats were pretreated for 4-5 days and then exposed to stressful stimulation. Basal hormone levels were not affected by the antagonists. In male rats, combined, but not separate blockade of NMDA and AMPA/kainate subtypes of glutamate receptors prevented the rise in plasma PRL in response to footshock stress. In female rats, footshock-induced PRL release was inhibited even by separate blockade of NMDA receptors by dizocilpine, suggesting that the PRL system of females is more sensitive to the effect of NMDA antagonists than that of males. None of the treatments affected PRL release during immobilization or ether stress. Simultaneous blockade of NMDA and AMPA receptor subtypes resulted in a mild inhibition of immobilization-induced ACTH release without any effect on ACTH response to footshock or ether stress. The data suggest that involvement of glutamatergic pathways in neuroendocrine response during stress is selective for discrete stress stimuli and stress hormones. In addition a concerted action of glutamate on both NMDA and non-NMDA receptor subtypes is involved in the control of PRL release during footshock stress.