Hypothalamo-pituitary-adrenocortical axis function and hedonic behavior in adult male and female rats prenatally stressed by maternal food restriction.

Neuroendocrine activation during stress is affected by many factors contributing to the variability of the stress response. The present study was aimed at evaluating long-term changes in hypothalamo-pituitary-adrenocortical (HPA) axis function and in hedonic behavior in adult offspring prenatally stressed by maternal food restriction, with attention on possible gender differences. Adult offspring were blood sampled via a tail artery cannula. Prenatally stressed females had significantly higher adrenal weights compared to males. Plasma ACTH levels, which rose in response to acute stress induced by handling, were significantly higher in females compared to those in males. A similar pattern was found in plasma corticosterone. The rise in ACTH levels was more pronounced in prenatally stressed rats though the rise in corticosterone failed to be modified. Corticotropin releasing hormone (CRH) and proopiomelanocortin mRNA levels in the hypothalamic paraventricular nucleus and anterior pituitary, respectively, were found to be unchanged. The present experiments failed to reveal a decrease in hedonic behavior in prenatally stressed rats. In contrast, in male offspring a tendency to a higher sucrose preference was observed. These data together with observed changes in hormone and CRH mRNA levels indicate that the gestational stress used did not result in a depression-like state in adult offspring.

Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain.

Apelin, a peptide recently isolated from bovine stomach tissue extracts, has been identified as the endogenous ligand of the human orphan APJ receptor. We established a stable Chinese hamster ovary (CHO) cell line expressing a gene encoding the rat apelin receptor fused to the enhanced green fluorescent protein, to investigate internalization and the pharmacological profile of the apelin receptor. Stimulation of this receptor by the apelin fragments K17F (Lys1-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) and pE13F (pGlu5-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) resulted in a dose-dependent inhibition of forskolin-induced cAMP production and promoted its internalization. In contrast, the apelin fragments R10F (Arg8-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) and G5F (Gly13-Pro-Met-Pro-Phe17) were inactive. The physiological role of apelin and its receptor was then investigated by showing for the first time in rodent brain: (i) detection of apelin neurons in the supraoptic and paraventricular nuclei by immunohistochemistry with a specific polyclonal anti-apelin K17F antibody; (ii) detection of apelin receptor mRNA in supraoptic vasopressinergic neurons by in situ hybridization and immunohistochemistry; and (iii) a decrease in vasopressin release following intracerebroventricular injection of K17F, or pE13F, but not R10F. Thus, apelin locally synthesized in the supraoptic nucleus could exert a direct inhibitory action on vasopressinergic neuron activity via the apelin receptors synthesized in these cells. Furthermore, central injection of pE13F significantly decreased water intake in dehydrated normotensive rats but did not affect blood pressure. Together, these results suggest that neuronal apelin plays an important role in the central control of body fluid homeostasis.

Corticotropin-releasing hormone mRNA levels in response to chronic mild stress rise in male but not in female rats while tyrosine hydroxylase mRNA levels decrease in both sexes.

Corticotropin-releasing hormone (CRH) and catecholamines are suggested to play a significant role in the pathophysiology of depression. In the present study we investigated gene expression of CRH in the hypothalamic paraventricular nucleus (PVN) and tyrosine hydroxylase (TH) in the locus coeruleus (LC) in an experimental model of depression. A chronic mild stress model was applied in rats of both genders for a three-week period. Anhedonic behaviour, a typical sign of depression-like state, was measured by a sucrose preference test. The chronic mild stress induced a decrease in sucrose preference in both genders. The body weight gain was reduced in males only. The total activity in the open field test was unchanged, however, male rats exposed to chronic mild stress showed enhanced locomotor activity during the first minute of the session, suggesting increased anxiety. Basal plasma corticosterone levels, thymus and adrenal weights measured on the third day after cessation of the stress regimen, were not affected by the stress procedure. Evaluation of CRH mRNA levels in the PVN by in situ hybridisation revealed a significant rise in response to chronic mild stress in males. In females, the basal CRH mRNA levels were higher compared to those in males, but the stress-induced rise was absent. Chronic mild stress resulted in a decrease in TH mRNA levels in the LC. These data demonstrate that chronic mild stress model of depression induces a specific stress response with a reduction of TH gene expression in the LC and clear gender differences in gain of body weight, anxiety-like behaviour, and CRH mRNA levels in the PVN.

Quinolinic acid enhances permeability of rat brain microvessels to plasma albumin.

Several studies have established that increased cerebrospinal fluid (CSF) levels of quinolinic acid (QUIN), a macrophage/microglia-derived excitotoxin with N-methyl-D-aspartate (NMDA)-receptor affinity, may reflect abnormal blood-brain barrier (BBB) function in patients with acquired immunodeficiency syndrome (AIDS) dementia complex, exhibiting a relationship to their clinical and neurological status. This study was aimed to evaluate whether QUIN (250 nmol/0.25 microl/ventricle) infused into both lateral cerebral ventricles permeates adult rat brain microvessels to plasma albumin. Possible BBB dysfunction was examined 4 days after the intracerebroventricular (i.c.v.) infusion of QUIN by measuring plasma albumin extravasation using rocket immunoelectrophoresis. The i.c.v. infusion of QUIN failed to increase the extracellular tissue concentration of albumin in the entorhinal cortex, but significantly higher levels were found in the hippocampus proper (but not in the subiculum region and dentate gyrus) and in the striatum. To evaluate the possible relationship between plasma protein extravasation and QUIN-induced tissue necrosis, we quantified neuronal death in the rat hippocampal formation (subiculum, CA1/CA3 areas of the hippocampus proper, dentate gyrus). We found significantly higher tissue levels of plasma albumin in the hippocampus proper, in which the CA1 area exhibited the highest neuronal loss while the low rate of neuronal death was not accompanied by significant albumin extravasation in the dentate gyrus. However, in case of the subiculum, in which the neuronal loss reached comparable values to those in the CA1 area, we did not find significant enhancement of plasma albumin leakage into this area. The regional differences in brain microvascular permeability may depend on the density of NMDA receptors in the multicellular capillary barrier, but the differences in neuronal death may also reflect an involvement of NMDA receptors in neuronal membranes. We conclude that increased CSF QUIN levels evoke a dysfunction of the BBB that may only partially be related to sites with pronounced neuronal damage in the rat brain regions susceptible to NMDA-receptor mediated toxicity.

Neonatal stress alters habituation of exploratory behavior in adult male but not female rats.

The effect of monosodium-L-glutamate (MSG) administration in the neonatal period on habituation of exploratory behavior related to gender differences was investigated. Rats of both sexes were intraperitoneally treated with MSG (4 mg/g) or hypertonic saline (10% NaCl) on postnatal days 2, 4, 6, 8, and 10. On postnatal day 65, the animals were tested in an open-field test during 4 consecutive days, once daily in 6-min sessions. The rapidity of habituation of exploratory behavior during repeated exposure to the open field (interrupted habituation) and over individual sessions (uninterrupted habituation) was evaluated by using the method of linear regression. Compared to intact controls, there were no significant differences found in interrupted habituation, neither in males nor in females. Uninterrupted habituation in neonatally treated males was slowed down in the first 2 days of testing. No differences in adult behavior between treated groups (MSG and hypertonic saline) were observed, i.e., there were no late effects specific for neonatal MSG administration. In females, uninterrupted habituation was not affected. Males proved to be more sensitive to neonatal stress associated with injections of MSG or hypertonic saline than females, and showed feminine-like habituation in the new environment.

Corticotropin-releasing hormone synthesizing neurons in the hypothalamic paraventricular nucleus of rats neonatally treated with monosodium glutamate can respond to different stress paradigms.

Neonatal administration of monosodium glutamate (MSG) produces pathological lesions in many brain regions. There are indications that MSG treatment could also influence the neurons of the hypothalamic paraventricular nucleus (PVN). The goal of this study was to find out whether MSG treatment could alter the activity of the corticotropin-releasing hormone synthesizing neurons, i.e. the principal regulators of the corticotropin hormone secretion, located in the medial posterior subdivision of the PVN. The activity of CRH neurons was assessed by changes in CRH mRNA levels in response to both stimulatory and inhibitory conditions induced by immobilization and water deprivation, respectively. In addition, effect of the circulating glucocorticoid deficit induced by bilateral adrenalectomy was investigated. The obtained data show that in MSG-treated animals the rise in CRH mRNA in response to immobilization stress and adrenalectomy as well as the decrease after water deprivation were similar to the changes seen in controls. In addition POMC mRNA changes in MSG-treated animals indicate an uninterrupted capability of CRH neurons to transform different signals to corticotropin cells. It can be concluded that CRH neurons of the PVN are not functionally altered, in spite of the widespread neurotoxic effect of MSG treatment.

Dissociation of changes in hypothalamic corticotropin-releasing hormone and pituitary proopiomelanocortin mRNA levels after prolonged stress exposure.

Repeated immobilization stress for 7 days caused marked increase in corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus (PVN, 90% above the control) and proopiomelanocortin (POMC) mRNA in the anterior pituitary (73% above the control) in rats. Prolongation of the stress exposure to 42 days led to a decline of CRH mRNA response (42% above the control) in comparison with that after 7 days, while POMC mRNA persisted at the same level. Observed adaptation of CRH but not POMC responses suggests that other factor(s) next to CRH participate in sustained rise in POMC expression during long-term stress.

Neurotoxic lesions induced by monosodium glutamate result in increased adenopituitary proopiomelanocortin gene expression and decreased corticosterone clearance in rats.

Hypothalamic-pituitary-adrenocortical function in rats with brain lesions induced by neonatal monosodium glutamate (MSG) treatment (4 mg/g, 5 administrations, i.p.) was evaluated in the present study. Using in situ hybridization we found increased proopiomelanocortin (POMC) mRNA levels in the adenopituitary and normal corticotropin-releasing hormone mRNA levels in the hypothalamic paraventricular nucleus in MSG-treated rats. The total content of pituitary adrenocorticotropin (ACTH) was not changed, while pituitary ACTH concentration was higher in MSG-treated compared to control rats. The number of ACTH-immunostained cells per a constant area of adenohypophysial section, as measured by immunohistochemistry, was unchanged indicating that no significant condensation of corticotropes occurred. Basal plasma ACTH concentrations were not different, whereas morning corticosterone levels were elevated in rats with MSG treatment. While ACTH response to stress stimuli was similar in both groups of rats, corticosterone response to exogenous ACTH (500 ng/kg, i.v., Synacthen), short-lasting handling and immobilization was of the same magnitude but prolonged in MSG-treated rats. Based on the decline of [3H] corticosterone in plasma, a decreased corticosterone clearance rate was found in MSG-treated rats. These findings suggest that MSG treatment results in increased POMC gene expression per corticotrope of the atrophic pituitary resulting in maintenance of normal pituitary ACTH stores and plasma ACTH levels. Elevated basal levels of corticosterone in plasma as well as prolonged corticosterone responses to stimulations in rats treated with MSG seem to be due to a decreased clearance rate of corticosterone.

Stress-induced increase in blood-brain barrier permeability in control and monosodium glutamate-treated rats.

Glutamate administration in neonatal rats causes reversible changes in blood-brain barrier (BBB) permeability and known neurotoxic lesions. This study was aimed to evaluate whether glutamate administered to neonatal rats influences properties of the developing BBB with consequences on adult BBB function. The vulnerability of the BBB was examined after short-lasting stress exposure by measurement of plasma albumin extravasation using immunoelectrophoresis. In control rats, 30 min of immobilization stress resulted in increased endogenous albumin extravasation in the hypothalamus, hippocampus, brain stem and cerebellum, but not in the cortex and striatum. Basal levels of albumin in adult glutamate-treated rats (4 mg monosodium glutamate/g BW, IP, five times during neonatal period) were significantly lower in the hypothalamus compared to that in controls. Stress-induced increase in albumin levels was lower in the brain stem, higher in the hypothalamus, and similar in other brain regions studied in glutamate-treated rats in comparison with controls. It is concluded that short-lasting immobilization stress increased BBB permeability in some but not all brain regions studied. Glutamate treatment of neonatal rats resulted in low basal albumin levels in the hypothalamus but did not exert a pronounced influence on adult BBB function. BBB vulnerability in glutamate-treated rats during stress exposure was increased in the hypothalamus and decreased in the brain stem.

Changes of exploratory behaviour and its habituation in rats neonatally treated with monosodium glutamate.

In an attempt to elucidate mechanisms involved in adaptation to a novel environment, consequences of neurotoxic damage induced by administration of monosodium glutamate (MSG) to both male (n = 42) and female (n = 45) rats in the early postnatal period were studied. Rats treated with MSG and appropriate controls were tested on postnatal days 21 and 65 for alterations of exploration and the rapidity of habituation changes in an open field test. Compared with intact animals, a high dose of MSG (4 mg/g) increased exploratory behaviour, with a subsequent decrease in the rapidity of habituation of male rats. Neonatal stress represented by hypertonic saline injection in a vehicle-control group induced a slight increase of exploratory behaviour as compared with intact animals. Males proved to be more vulnerable to neonatal MSG treatment and handling than females. These results suggest a negative effect of neonatal stress and treatment with MSG on habituation to a new environment in male rats.

Neuroendocrine response during stress with relation to gender differences.

Neuroendocrine activation belongs to the main characteristics of the stress response. This response is not uniform but depends on the stress stimulus involved and on many other factors including the gender of the individual. In rats, corticosterone and ACTH levels as well as functional activity of the hypothalamo-pituitary-adrenocortical axis are higher in females compared to males under both basal and stress conditions. Marked sex differences were observed in stress-induced changes posterior pituitary hormone release. In male rats, release of vasopressin is not stimulated during stress conditions without an osmotic component while in female rats a rise in plasma vasopressin levels was observed even after short immobilization. Oxytocin release is enhanced in response to the majority of stress stimuli and it was found to be greater in females than in males. Mentioned gender differences are attributed to the effect of sex steroids, particularly those of estrogens. Not enough information is available on gender differences in the neuroendocrine response during stress in humans. We observed a greater neuroendocrine activation in women than in men in response to heat exposure in sauna with pronounced differences in ACTH and prolactin release and partly also after a cold-pressor test. Understanding of gender differences in neuroendocrine response during stress might contribute to the explanation of the development of some emotional and other disorders with higher incidence in women.

Vasopressin and oxytocin in stress.

Though oxytocin and vasopressin are similar in structure and are produced in the same brain regions, they show specific responses under stress conditions. In humans, increases in peripheral blood vasopressin appear to be a consistent finding during many acute stress situations, while in rats, vasopressin secretion is unresponsive to several stimuli known to induce ACTH and catecholamine release. Even decreases in vasopressin levels during stress were described. In accordance with others, we observed enhanced vasopressin release in response to stress stimuli with an osmotic component such as hypertonic saline injection but also during exposure of rats to a warm environment. Immobilization stress which fails to induce vasopressin release was reported to increase hypothalamic vasopressin mRNA and plasma vasopressin levels in chronically adreno-demedullated rats. Unlike vasopressin, oxytocin may be considered a typical stress hormone responding to osmotic as well as other stress stimuli. We found that acute exposure of rats to immobilization stress resulted in an increase in oxytocin mRNA level. In addition, we have shown that magnocellular neurons of the paraventricular nucleus, but not the supraoptic nucleus, are essential for oxytocin release during immobilization stress. The release of posterior pituitary hormones represents an important component of the stress response.

Albumin content in the developing rat brain in relation to the blood-brain barrier.

The problem of functional development of the blood-brain barrier (BBB) has been the subject of intensive discussion for many years. Some morphological data indicate that the cerebral endothelium becomes impermeable to macromolecules already before birth. On the other hand, specific features in transport of macromolecular markers in fetuses and neonates were described supporting the hypothesis of an immature BBB. Part of the confusion seems to result from inadequately designed experiments, concerning frequently the administration of markers. In our study we decided not to use an exogenous marker and to follow up to changes of endogenous albumin content in selected brain regions of rats from the neonatal period to adulthood. Shortly after birth, the concentrations of albumin in all brain regions studied (hypothalamus, hippocampus, cortex, striatum, brainstem, cerebellum) were significantly higher compared to those of adult animals. Moreover, the distribution of albumin within these structures in newborns differed from that in adults. Several factors may contribute to the accumulation of albumin in the developing rat brain, including increased permeability of the BBB.