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Gulf War Illness (GWI) is a multi-symptom illness that continues to affect over 250,000 American Gulf War veterans. The causes of GWI remain equivocal; however, prophylactic use of the acetylcholinesterase inhibitor pyridostigmine bromide (PB), and the stress of combat have been identified as two potential causative factors. Both PB and stress alter acetylcholine (ACh), which mediates both cognition and anti-inflammatory responses. As inflammation has been proposed to contribute to the cognitive deficits and immune dysregulation in GWI, the goal of this study was to determine the long-term effects of PB and stress on the cholinergic anti-inflammatory pathway in the central nervous system and periphery. We used our previously established rat model of GWI and in vivo microdialysis to assess cholinergic neurochemistry in the prefrontal cortex (PFC) and hippocampus following a mild immune challenge (lipopolysaccharide; LPS). We then examined LPS-induced changes in inflammatory markers in PFC and hippocampal homogenates. We found that PB treatment produces a long-lasting potentiation of the cholinergic response to LPS in both the PFC and hippocampus. Interestingly, this prolonged effect of PB treatment enhancing cholinergic responses to LPS was accompanied by paradoxical increases in the release of pro-inflammatory cytokines in these brain regions. Collectively, these findings provide evidence that neuroinflammation resulting from dysregulation of the cholinergic anti-inflammatory pathway is a mechanistic mediator in the progression of the neurochemical and neurocognitive deficits in GWI and more broadly suggest that dysregulation of this pathway may contribute to neuroinflammatory processes in stress-related neurological disorders., Competing Interests: The authors declare no competing financial interests., (© 2022 The Authors.)

Insulin resistance is a major contributor to the neuroplasticity deficits observed in patients with metabolic disorders. However, the relative contribution of peripheral versus central insulin resistance in the development of neuroplasticity deficits remains equivocal. To distinguish between peripheral and central insulin resistance, we developed a lentiviral vector containing an antisense sequence selective for the insulin receptor (LV-IRAS). We previously demonstrated that intra-hippocampal injection of this vector impairs synaptic transmission and hippocampal-dependent learning and memory in the absence of peripheral insulin resistance. In view of the increased risk for the development of neuropsychiatric disorders in patients with insulin resistance, the current study examined depressive and anxiety-like behaviors, as well as hippocampal structural plasticity in rats with hippocampal-specific insulin resistance. Following hippocampal administration of either the LV-control virus or the LV-IRAS, anhedonia was evaluated by the sucrose preference test, despair behavior was assessed in the forced swim test, and anxiety-like behaviors were determined in the elevated plus maze. Hippocampal neuron morphology was studied by Golgi-Cox staining. Rats with hippocampal insulin resistance exhibited anxiety-like behaviors and behavioral despair without differences in anhedonia, suggesting that some but not all components of depressive-like behaviors were affected. Morphologically, hippocampal-specific insulin resistance elicited atrophy of the basal dendrites of CA3 pyramidal neurons and dentate gyrus granule neurons, and also reduced the expression of immature dentate gyrus granule neurons. In conclusion, hippocampal-specific insulin resistance elicits structural deficits that are accompanied by behavioral despair and anxiety-like behaviors, identifying hippocampal insulin resistance as a key factor in depressive illness., Competing Interests: The authors declare no competing financial interests., (© 2021 The Authors.)

Major depressive illness is among the most prevalent neuropsychiatric disorders and is associated with neuroplasticity deficits in limbic structures such as the amygdala. Since exposure to stressful life events is proposed to contribute to depressive illness, our recent studies examined the effects of stress on amygdalar neuroplasticity. These studies determined that repeated stress elicits deficits in glutamatergic activity in the amygdala, neuroplasticity deficits that can be prevented by some but not all antidepressants. In view of these observations, the goal of the current study was to determine the effects of repeated restraint stress (RRS) on the dendritic architecture of pyramidal neurons in the rat basolateral nucleus of the amygdala (CBL), as well as glutamate efflux in the CBL and central nucleus of the amygdala (CMX) via in vivo microdialysis. We also examined the ability of the antidepressant agomelatine to prevent RRS-induced neuroplasticity deficits. Compared with control rats, rats subjected to RRS exhibited atrophy of CBL pyramidal neurons, including decreases in total dendritic length, branch points, and dendritic complexity index. In addition, glutamate efflux was significantly reduced in the CMX of rats subjected to RRS, thereby identifying a potential neurochemical consequence of stress-induced dendritic atrophy of CBL pyramidal neurons. Lastly, an acute stress challenge increased corticosterone (CORT) levels in the CBL, suggesting that stress-induced increases in CORT levels may contribute to the neuroanatomical and neurochemical effects of RRS in the CBL. Importantly, these RRS-induced changes were prevented by daily agomelatine administration. These results demonstrate that the neuroanatomical and neurochemical properties of glutamatergic neurons in the rat amygdala are adversely affected by repeated stress and suggest that the therapeutic effects of agomelatine may include protection of structural and neurochemical plasticity in limbic structures like the amygdala., (Published by Elsevier Ltd.)

In the central nervous system (CNS) insulin mediates a variety of effects including feeding, metabolism and cognition. The cognitive enhancing effects of insulin are proposed to be mediated through activation of insulin receptors in the hippocampus, an important integration center for learning and memory in the mammalian brain. Since less is known regarding insulin signaling events in the hippocampus, the aim of the current study was to determine whether insulin stimulates similar signaling cascades and GLUT4 translocation in the rat hippocampus as has been described in peripheral tissues. Intracerebroventricular administration of insulin increases hippocampal insulin levels and also stimulates the phosphorylation of Akt in a time-dependent manner. Insulin also stimulates the translocation of GLUT4 to hippocampal plasma membranes in a time course that mirrors the increases in glucose uptake observed during the performance of hippocampal-dependent tasks. Insulin stimulated phosphorylation of Akt and translocation of GLUT4 were blocked by pretreatment with the PI3-kinase inhibitor LY294002. Confocal immunofluorescence determined that insulin stimulated phosphorylation of Akt was localized to neurons and colocalized with the insulin receptor and GLUT4 in the rat hippocampus, thereby identifying the functional anatomical substrates of insulin signaling in the hippocampus. These results demonstrate that insulin-stimulated translocation of GLUT4 to the plasma membrane in the rat hippocampus occurs via similar mechanisms as described in peripheral tissues and suggests that insulin-mediated translocation of GLUT4 may provide a mechanism through which hippocampal neurons rapidly increase glucose utilization during increases in neuronal activity associated with hippocampal-dependent learning.

Regulation of feeding behavior and energy balance are among the central effects of insulin. For example, intracerebroventricular administration of insulin decreases food intake and body weight, whereas antisense oligodeoxynucleotide downregulation of insulin receptors (IRs) produces hyperphagia. To further examine the role of IRs in the central actions of insulin, we designed an IR antisense lentiviral vector (LV-IRAS) and injected this vector into the third ventricle to selectively decrease IR expression in the rat hypothalamus. Three weeks after LV-IRAS administration, the expression of IRs in the hypothalamus was significantly decreased, whereas no changes were observed in hippocampal IR levels. LV-IRAS administration decreased insulin-stimulated phosphorylation of hypothalamic IRs and translocation of the insulin-sensitive glucose transporter GLUT4 in the hypothalamus; no changes in IR signaling were observed in the hippocampus of LV-IRAS-treated rats. Lentivirus-mediated downregulation of IR expression and signaling produced significant increases in body weight, as well as increases in fat mass that were selective for the subcutaneous compartment. Conversely, lean muscle mass and water mass were not affected in LV-IRAS-treated rats compared to rats treated with control virus. Changes in peripheral adiposity were associated with increases in basal hypothalamic leptin signaling in the absence of changes in leptin receptor expression in LV-IRAS rats. Collectively, these data illustrate the important functional relationships between hypothalamic insulin and leptin signaling in the regulation of body composition and provide insight into the mechanisms through which decreases in IR expression and signaling dysregulates leptin activity, thereby promoting increases in peripheral adiposity.

The hippocampus, an important integration center for learning and memory in the mammalian brain, undergoes neurological changes in response to a variety of stimuli that are suggestive of ongoing synaptic reorganization. Accordingly, the aim of this study was to identify markers of synaptic plasticity using rapid and reliable techniques such as radioimmunocytochemistry and confocal microscopy, thereby providing a "birds-eye view" of the whole hippocampus under hypercorticosteronemic conditions. The regulation of microtubule-associated protein 2, synaptophysin and postsynaptic density-95 was examined in two different animal models of hypercorticosteronemia: corticosterone administration and streptozotocin-induced diabetes using both a short-term (1 week) and long-term (5 weeks) treatment. Glucocorticoids and/or hyperglycemia increased synaptophysin expression in CA1, CA3 and the dentate gyrus, regions that exhibit synaptic plasticity in response to glucocorticoid exposure. In these models, postsynaptic density-95 expression increased in the CA3 region, particularly in the diabetic rats, while microtubule-associated protein 2 exhibited more selective changes. Fluoro-Jade histochemistry did not detect neuronal damage, suggesting that glucocorticoids and/or hyperglycemia induce plastic and not irreversible neuronal changes at these time points. Collectively, these results demonstrate that changes in the expression and distribution of synaptic proteins provide another measure of synaptic plasticity in the rat hippocampus in response to glucocorticoid exposure, changes that may accompany or contribute to neuroanatomical, neurochemical, and behavioral changes observed in experimental models of type 1 diabetes.

Oxidative stress and modulation of anti-oxidant enzymes may contribute to the deleterious consequences of diabetes mellitus and to the effects of chronic (i.e. 21 day) stress in the CNS. We therefore compared the effects of short- and long-term exposure to diabetes-induced hyperglycemia, restraint stress and the combined effects of restraint stress and diabetes upon parameters of oxidative stress in the rat hippocampus. Whereas 7 days of restraint stress or hyperglycemia, or the combination, produced similar increases in oxidative stress markers 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) throughout the hippocampus, 21 days of stress or hyperglycemia did not increase these markers in the dentate gyrus. In contrast, Ammon's horn still showed elevated levels of these lipid peroxidation products, especially in diabetic rats subjected to 21 days of restraint stress. The expression of two anti-oxidant enzymes, copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese SOD, was also differentially regulated by stress and hyperglycemia in a time- and region-specific manner in the rat hippocampus. Although long-term stress decreased both SOD isoforms, diabetes increased Cu/Zn-SOD expression in DG with or without 21 days of repeated stress. These increases may account for the finding that protein-conjugated HNE and MDA levels returned to control levels between 7 days and 21 days of hyperglycemia or the combination of diabetes and stress. These results suggest that while other anti-oxidant pathways may account for decreases in oxidative stress in the long-term stress paradigm, increases in Cu/Zn-SOD expression may contribute to the region-specific attenuation of oxidative stress in the diabetic rat hippocampus.

1. Previous work demonstrated that estradiol (E2) treatment prevented the abnormal response to stress and the reduction of glucocorticoid receptors (GR) in hippocampus from aging male rats. The mechanisms originating these effects were unknown. 2. In the present work, we investigated the E2 effects on the cholinergic, growth-associated protein (GAP-43) expressing neurons of the medial septum (MS) and vertical limb of diagonal band of Broca (VDB). These areas project to the hippocampus, and may be involved in the mentioned E2 effects in aging animals. Therefore, the response to E2 of choline-acetyltransferase (ChAT) in neurons and cell processes and GAP-43 mRNA as a marker of neurite outgrowth was studied in young and old male rats. 3. Young (3-4 months) and old (18-20 months) male Sprague-Dawley rats remained untreated or were implanted s.c. with a 14 mg pellet of E2 benzoate during 6 weeks. We used immoucytochemistry to determine ChAT and isotopic in situ hybridization to analyze GAP-43 mRNA expression. 4. Aging males showed a reduction in the number and length of ChAT-immunoreactive cell processes, but not in the number of positive neurons in MS and VDB. E2 reverted both parameters in old rats to levels of young animals. Regarding basal levels of GAP-43 mRNA, they were similar in old and young animals, but E2 treatment up-regulated GAP-43 mRNA expression in MS and VDB of old animals only. 5. Our data suggest that prolonged E2 treatment may affect hippocampal function of aging male rats by regulating in part the plasticity of cholinergic, GAP-43 expressing neurones of the basal forebrain. Without discarding a direct E2 effect on the limbic tissue, effects on the cholinergic system may have a pronounced impact on the neuroendocrine and stress responses of the aging hippocampus.

We describe the localization of the recently identified glucose transporter GLUTx1 and the regulation of GLUTx1 in the hippocampus of diabetic and control rats. GLUTx1 mRNA and protein exhibit a unique distribution when compared with other glucose transporter isoforms expressed in the rat hippocampus. In particular, GLUTx1 mRNA was detected in hippocampal pyramidal neurons and granule neurons of the dentate gyrus as well as in nonprincipal neurons. With immunohistochemistry, GLUTx1 protein expression is limited to neuronal cell bodies and the most proximal dendrites, unlike GLUT3 expression that is observed throughout the neuropil. Immunoblot analysis of hippocampal membrane fractions revealed that GLUTx1 protein expression is primarily localized to the intracellular compartment and exhibits limited association with the plasma membrane. In streptozotocin diabetic rats compared with vehicle-treated controls, quantitative autoradiography showed increased GLUTx1 mRNA levels in pyramidal neurons and granule neurons; up-regulation of GLUTx1 mRNA also was found in nonprincipal cells, as shown by single-cell emulsion autoradiography. In contrast, diabetic and control rats expressed similar levels of hippocampal GLUTx1 protein. These results indicate that GLUTx1 mRNA and protein have a unique expression pattern in rat hippocampus and suggest that streptozotocin diabetes increases steady-state mRNA levels in the absence of concomitant increases in GLUTx1 protein expression.

Female F344 rats treated chronically with diethylstilbestrol (DES) develop prolactin (PRL)-producing pituitary tumors. These tumors are larger in female than in male rats. To investigate gender differences in DES-induced pituitary tumor formation, we employed female and male rats and neonatally androgenized females, which received 100 microg of testosterone propionate (TP) after birth. At 3 months of age, all rats were deprived of their gonads and divided into control and DES-treated groups. Forty days after beginning treatment, control pituitary weight and serum PRL were similar in gonadectomized males (GDX), ovariectomized females (OVX) and androgenized-ovariectomized females (OVX + TP), but weight of DES-induced tumors was 2.5-fold higher and serum PRL 5.6-fold higher in OVX + DES than in GDX + DES or OXV + TP + DES (p<0.001). At the pituitary level, nuclear estrogen receptors (NE(2)R) amounted to >100 fmol/mg DNA in all rats receiving DES. However, NE(2)R were lower in OVX + DES (101.3+/-9.0 fmol/mg DNA) than in GDX + DES (174.6 +/-16.8; p<0.05) and in OXV + DES + TP (150.3+/-27.7; p<0.05). A similar profile was found for cytosolic progestin receptors. Using electron microscopy (EM), hyperplasia/hypertrophy of lactotropes was found in all DES-stimulated pituitaries. However, tumors of OVX + DES rats were enriched in hyperstimulated typical lactotropes, i.e., cells with high rate of hormonal synthesis, processing and secretion. Instead, tumors from GDX + DES and OVX + TP + DES rats were a mixture of typical and atypical lactotropes, i.e. a cell subpopulation with refractory secretory response and a few gonadotropes. In agreement with these data, immunoreactive pituitary PRL was lower in OVX + DES than in OVX + TP + DES and GDX + DES groups. Thus, differences in the sensitivity to DES, serum and tumor PRL, NE(2)R and progestin receptors between estrogenized female rats on one side and male and TP-androgenized females on the other, may by due in part to heterogeneity of cell populations. Our data further suggest that neonatal hypothalamic exposure to androgens, as in normal males or androgenized females with masculinization of hypothalamic centers, may condition the response to DES stimulation later in life., (Copyright 2000 S. Karger AG, Basel.)

Hexachlorobenzene (HCB) is a polyhalogenated aromatic hydrocarbon widely distributed in the environment. In animal testing, HCB has been shown to be a reproductive toxin. Previous investigations of the effects of HCB on ovarian function have yielded equivocal results. Thus, the effects of chronic administration of HCB (1 g kg(-1) body wt.) on the ovary and pituitary hormone levels, hepatic and uterine oestradiol receptors, ovarian histopathological changes and oestrus cycle characteristics were investigated in spontaneously cycling rats. Our data demonstrate that HCB treatment, under the conditions of the present study, reduced circulating levels of oestradiol and prolactin without differences in serum concentrations of progesterone. Follicle-stimulating hormone serum levels were elevated. Hexachlorobenzene treatment resulted in irregularity of cycles, characterized mainly as prolonged periods of oestrus with a reduced number of ova recovered. In addition, HCB administration resulted in significantly decreased uterine nuclear oestrogen receptor levels. Histopathological examination revealed degenerative changes of the ovarian follicles and germinal epithelium and increased numbers of atresic follicles., (Copyright 2000 John Wiley & Sons, Ltd.)

Aging is associated with a disturbance in the regulation of the hypothalamic-pituitary-adrenal axis (HPA) and reduced levels of glucocorticoid receptors (GR) in the hippocampus. To compensate for these effects, we have investigated whether estrogen therapy normalized the HPA response to stress and GR in hippocampus and paraventricular (PVN) nucleus. Young (3-4 months) and old (20 months) male Sprague-Dawley rats were bled by tail cut in the basal state and following ether stress. While basal and ether-stimulated levels of plasma corticosterone (CORT) were similar in the two groups, old animals presented a delayed termination of the response to ether stress. A dexamethasone inhibition test carried out in old animals, showed a failure to completely block plasma CORT after ether stimulation. Furthermore, in old rats GR-immunoreactive levels were reduced in CA1-CA2 hippocampal subfields and subiculum, while normal levels were obtained in CA3-CA4 and PVN. We observed that prolonged estrogen treatment (6 weeks) of old rats normalized the termination of the stress response, restored dexamethasone inhibition of plasma CORT, and increased GR immunoreactivity in CA1 and CA2 hippocampal subfields and subiculum. The results suggest that estrogen treatment enhanced the glucocorticoid feedback signal by increasing GR in hippocampus, and corrected the disturbances in HPA axis regulation. These animal experiments may be important to elucidate the effects of estrogenic on the hippocampal and HPA dysfunction associated with aging and Alzheimer's disease in humans.

The neuropeptides arginine vasopressin (AVP) and oxytocin (OT) have been implicated in the genesis of hypertension due to deoxycorticosterone acetate (DOCA)-salt treatment of uninephrectomized rats. In this work, we studied if DOCA treatment of intact rats in doses arousing a salt appetite (a prehypertensive state), modulated mRNA for AVP and OT in the hypothalamus. Male Sprague-Dawley rats were offered both tap water and 3% NaCl in separate bottles and received vehicle or subcutaneous injections of 10 mg DOCA on alternate days for 7 days (4 injections) or 17 days (9 injections). They developed a preference for 3% NaCl solutions 24-48 h after treatment. Brain slices from rats killed on the 8th or 18th day were exposed to 35S-labeled probes encoding prepro-AVP mRNA or OT mRNA, respectively. Expression of these mRNAs was measured in the magnocellular and parvocellular divisions of the paraventricular nucleus (PVN) and magnocellular cells of the supraoptic nucleus (SON). No changes were obtained in neuropeptide mRNA levels in the parvocellular division of the PVN between control and the two groups of DOCA-treated rats. However, DOCA-treated animals presented an increased number of grains per cell for AVP mRNA in the magnocellular division of the PVN and in magnocellular cells of the SON, as shown by group mean comparisons and frequency histograms. No changes were detected for OT mRNA. In a second series of studies, control or DOCA-treated rats were offered 3% NaCl or water as the only choice. Animals drinking 3% NaCl showed increased AVP and OT mRNA levels, whether they received DOCA or not. However, AVP mRNA levels in both nuclei were higher in DOCA-treated rats drinking 3% NaCl than in controls drinking salt solution. In comparison, control and DOCA-treated rats drinking water showed lower levels of AVP mRNA. OT mRNA levels in the SON remained unchanged in the same groups. The results suggest that in the magnocellular cells of the PVN and SON, increments in AVP mRNA are obtained following increments in salt intake produced by either mineralocorticoid treatment or exclusive salt drinking. In rats offered salt solution and water to drink, DOCA effects on AVP mRNA developed before changes occurred in serum sodium levels. Because combined DOCA + salt treatment induced a higher response in terms of AVP mRNA expression, we suggest that AVP could be a target of the central effects of the mineralocorticoid.

Glucocorticoids (GC) and mineralocorticoids (MC) have profound regulatory effects upon the central nervous system (CNS). Hormonal regulation affects several molecules essential to CNS function. First, evidences are presented that mRNA expression of the alpha3 and beta1-subunits of the Na,K-ATPase are increased by GC and physiological doses of MC in a region-dependent manner. Instead, high MC doses reduce the beta1 isoform and enzyme activity in amygdaloid and hypothalamic nuclei, an effect which may be related to MC control of salt appetite. The alpha3-subunit mRNA of the Na,K-ATPase is also stimulated by GC in motoneurons of the injured spinal cord, suggesting a role for the enzyme in GC neuroprotection. Second, we provide evidences for hormonal effects on the expression of mRNA for the neuropeptide arginine vasopressin (AVP). Our data show that GC inhibition of AVP mRNA levels in the paraventricular nucleus is sex-hormone dependent. This sexual dimorphism may explain sex differences in the hypothalamic-pituitary-adrenal axis function between female and male rats. Third, steroid effects on the astrocyte marker glial fibrillary acidic protein (GFAP) points to a complex regulatory mechanism. In an animal model of neurodegeneration (the Wobbler mouse) showing pronounced astrogliosis of the spinal cord, in vivo GC treatment down-regulated GFAP immunoreactivity, whereas the membrane-active steroid antioxidant U-74389F up-regulated this protein. It is likely that variations in GFAP protein expression affect spinal cord neurodegeneration in Wobbler mice. Fourth, an interaction between neurotrophins and GC is shown in the injured rat spinal cord. In this model, intensive GC treatment increases immunoreactive low affinity nerve growth factor (NGF) receptor in motoneuron processes. Because GC also increases immunoreactive NGF, this mechanism would support trophism and regeneration in damaged tissues. In conclusion, evidences show that some molecules regulated by adrenal steroids in neurons and glial cells are not only involved in physiological control, but additionally, may play important roles in neuropathology.

1. Arginine vasopressin (AVP) is synthesized in specific brain regions including the magnocellular and parvocellular divisions of the paraventricular nucleus (PVN). Whereas magnocellular AVP responds to osmotic stimuli and functions mainly--although not exclusively--as an antidiuretic hormone, that produced in the parvocellular region controls the hypothalamus-pituitary-adrenal (HPA) axis, in conjunction with CRF. 2. In view of the reported sex differences in control of the HPA axis, we studied if these also pertain to AVP mRNA in the PVN of ovariectomized-estrogenized female rats and male rats determined by in situ hybridization. AVP mRNA was measured in intact rats, adrenalectomized (ADX) rats and ADX receiving dexamethasone (DEX) of both sexes. 3. Computerized autoradiography showed that in both sexes, AVP mRNA levels in the parvocellular division of the PVN increased after adrenalectomy and decreased following DEX. However, the reduction by DEX was more pronounced in female rats. No changes were found for the magnocellular region. Grain counting analysis of the medial-medial (MMP) and medial-lateral (MLP) subdivisions of the parvocellular region showed that the average number of grains per cell area in the MMP region of adrenally intact female rats was higher than that in males. However, in females there was no clear-cut effect of adrenalectomy on AVP mRNA levels, although the reduction after DEX treatment was again greater than that in male rats. Frequency histograms constructed by plotting the number of cells vs the number of grains per area substantiated the enhanced glucocorticoid negative control of AVP mRNA in the MMP and MLP of female rats. 4. The results indicated a sexual dimorphism in the glucocorticoid-dependent plasticity of AVP mRNA levels in the PVN. Because AVP mRNA expression differs between sexes under basal levels, after adrenalectomy, and after DEX treatment, these plastic changes may differentially condition the response to stress. Taking into consideration that stress and AVP may play a role in neurogenic hypertension, the possibility of sexual dimorphisms in AVP control may be important to assess the role of sex hormones in stress and steroid-derived hypertension.

In physiological doses, mineralocorticoids (MC) normalize the high salt intake developed after adrenalectomy. We have studied whether this effect of MC is accompanied by changes in the mRNA of neuronal alpha3 and beta1 subunits of the (Na,K)-ATPase because this enzyme could by a mediator of MC action in target cells. We employed [35S]oligonucleotide probes for the mentioned subunits hybridized to brain sections from adrenalectomized rats and adrenalectomized rats receiving aldosterone (ALDO) during 4 days. Using t-test statistics to measure differences in mean levels of grain density, and the Kolmogorov-Smirnov non-parametric test applied to frequency histograms, we showed that ALDO increased the alpha3 subunit mRNA in the septum medialis, preoptic area medialis, caudate-putamen, periventricular gray substance, amygdala lateralis, hippocampal subfields CA1 to CA4 and the gyrus dentatus. Significant increases for the beta1 subunit mRNA were found in periventricular gray substance, the CA1-CA4 hippocampal subfields and gyrus dentatus. Therefore, the salt-suppression effect of MC was accompanied by coordinate increases in (Na,K)-ATPase alpha3 and beta1 subunit mRNA in the hippocampus, gyrus dentatus and periventricular gray substance, whereas in other regions the stimulatory effect was exclusive of the alpha3 subunit mRNA only. The results suggest that the enzyme could be a target of ALDO action not only in areas related to salt appetite control (amygdala, preoptic area) but also in brain regions subserving other functions of the MC.

We have studied glucocorticoid receptors (GR) and actions in the spinal cord of the Wobbler mouse, a model for amyotrophic lateral sclerosis and infantile spinal muscular atrophy. Basal and stress levels of circulating corticosterone (CORT) were increased in Wobbler mice. Single point binding assays showed that cytosolic type II GR in the spinal cord of Wobbler mice of both sexes were slightly reduced compared with normal littermates. Saturation analysis further demonstrated a non-significant reduction in Bmax with increased Kd. In the hippocampus, however, we found down-regulation of GR, a probable response to increased CORT levels. We also found that the basal activity of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, was higher in Wobbler mice than in control animals. Both groups showed a two-fold stimulation of ODC activity after treatment with dexamethasone (DEX). Additionally, Wobbler mice presented with an intense proliferation of astrocytes immunoreactive (ir) for glial fibrillary acidic protein (GFAP) in grey and white matter of the spinal cord. The enhanced GFAP-ir was attenuated after four days of treatment with a corticosterone (CORT) pellet implant, producing a pharmacological increase in peripheral circulating CORT. Taking into consideration the content of GR and the changes in ODC activity and GFAP-ir brought about by glucocorticoids, we suggest that Wobbler mice are hormone responsive. Further elucidation of glucocorticoid effects in this model may be relevant for understanding the possible use of hormones in human neurodegenerative diseases.

It is known that chronic exposure of F344 rats to diethylstilbestrol (DES) induces prolactin (PRL)-secreting pituitary tumors composed of proliferating mammotropic cells. In the present work, we studied the effects of progesterone (P4) on several parameters stimulated in the pituitary tumors (DES-T), such as nuclear estrogen receptors (NE2R), cytosolic progestin receptors (CP4R) and serum PRL. Additionally, we have measured in hypothalamus the mRNA levels for tyrosine hydroxylase (TH), the rate-limiting enzyme for synthesis of dopamine, the main PRL-inhibitory factor. We found that pellet implantation of P4 during 1 month significantly reduced weight, ligand binding to NE2R and CP4R and serum PRL in the tumorous glands. Reductions in sex steroid receptor binding were due to changes in Bmax without changes in Kd, as observed after Scatchard plot analysis. Receptor binding data, therefore, suggests a pituitary site of action of P4. TH mRNA expression was studied in tuberoinfundibular dopaminergic (TIDA) neurons by in situ hybridization techniques employing a 35S-labeled oligonucleotide probe. Mean number of grains/cell decreased significantly in DES-T, an effect partly reversed by P4 treatment. Frequency histograms were constructed by plotting the number of cells versus the number of grains/cell and examined by x2 test and analysis of residuals. We found that DES-T presented significantly more cells with less grains whereas in control glands, P4-treated rats and DES-T receiving P4, cells with a higher grain number prevailed. These results suggest that in addition to a direct pituitary effect, P4 may also antagonize DES-induced tumorigenesis acting on mRNA for TH and presumably on the activity of TIDA neurons of the hypothalamus. The use of DES-T as a model for hyperprolactinemia may allow further assessment of P4 effects in pituitary adenomas in humans.

1. Wobbler mice suffer an autosomal recessive mutation producing severe motoneuron degeneration and dense astrogliosis, with increased levels of glial fibrillary acidic protein (GFAP) in the spinal cord and brain stem. They have been considered animal models of amyotrophic lateral sclerosis and infantile spinal muscular atrophy. 2. Using Wobbler mice and normal littermates, we investigated the effects of the membrane-active steroid Lazaroid U-74389F on the number of GFAP-expressing astrocytes and glucocorticoid receptors (GR). Lazaroids are inhibitors of oxygen radical-induced lipid peroxidation, and proved beneficial in cases of CNS injury and ischemia. 3. Four days after pellet implantation of U-74389F into Wobbler mice, hyperplasia and hypertophy of GFAP-expressing astrocytes were apparent in the spinal cord ventral and dorsal horn, areas showing already intense astrogliosis in untreated Wobbler mice. In control mice, U-74389F also produced astrocyte hyperplasia and hypertophy in the dorsal horn and hyperplasia in the ventral-lateral funiculi of the cord. 4. Given in vivo U-74389F did not change GR in spinal cord of Wobbler or control mice, in line with the concept that it is active in membranes but does not bind to GR. Besides, U-74390F did not compete for [3H]dexamethasone binding when added in vitro. 5. The results suggest that stimulation of proliferation and size of GFAP-expressing astrocytes by U-74389F may be a novel mechanism of action of this compound. The Wobbler mouse may be a valuable animal model for further pharmacological testing of glucocorticoid and nonglucocorticoid steroids in neurodegenerative diseases.

The Na+,K(+)-ATPase plays a key role in the regulation of ion fluxes and membrane repolarization in the CNS. We have studied glucocorticoid effects on biosynthesis of the Na+,K(+)-ATPase and on ouabain binding in the ventral horn of the spinal cord using intact rats, adrenalectomized (ADX) rats, and ADX rats receiving dexamethasone (ADX+DEX) during 4 days. Cryostat sections from spinal cords were incubated with a 35S-oligonucleotide coding for the alpha 3-subunit or a 3H-cDNA coding for the beta 1-subunit of the Na+,K(+)-ATPase using in situ hybridization techniques. In ventral horn motoneurons, grain density per cell and grain density per area of soma for both probes were slightly reduced in ADX rats but significantly increased in the ADX+DEX group, using ANOVA and the Bonferroni's test. Statistical analysis of frequency histograms of neuronal densities further indicated a significant shift to the right for intact rats compared with ADX rats for both probes. Concomitantly, [3H]ouabain binding to membrane preparations from ventral horns was reduced in ADX rats and restored to normal by DEX administration. No effect of adrenalectomy or DEX treatment was obtained in the dorsal horn. In conclusion, glucocorticoids positively modulate the mRNA for the alpha 3-subunit and the beta 1-subunit of the Na+,K(+)-ATPase and recover ouabain binding to normal values. The increments of the synthesis and activity of an enzyme affecting membrane repolarization and synaptic neurotransmission are consistent with the alleged stimulatory effect of glucocorticoids on spinal cord function.

The effect of glucocorticoids on (Na+K)ATPase mRNA synthesis was studied in 19 brain areas of adrenalectomized (ADX) rats untreated or receiving dexamethasone (DEX). For in situ hybridization, we employed a [35S]oligonucleotide probe for the alpha 3-subunit isoform, and a [3H]cDNA coding for the beta 1-subunit of the enzyme. Mean levels of grain density for the alpha 3 subunit mRNA of DEX-treated rats were significantly higher by a 't' test in medial septum, amygdala lateralis (AL) and medialis (AME), gyrus dentatus, CA4 hippocampal area, substantia nigra and periventricular gray, compared to untreated rats. For the beta 1-subunit, mean levels after DEX were significantly higher in AL and lateral preoptic area. In addition, the Kolmogorov-Smirnov test applied to frequency histograms of neuronal densities indicated a coordinate increase in alpha 3 and beta 1-subunit mRNA expression for the CA2 subfield and preoptic area medialis (POA MED). We conclude that (1) glucocorticoids are positive modulators of (Na+K)ATPase mRNA; (2) analysis of frequency histograms suggests that glucocorticoids promote in a few regions (AL, POA MED, CA2 subfield) a coordinate increase in the biosynthesis of the alpha 3 and beta 1-subunit mRNA. In 11 other areas stimulation occurs for one subunit mRNA only, whereas 5 areas were insensitive to glucocorticoid effects on this enzyme.

We previously reported a reduction of glucocorticoid receptors (GCR) in diethylstilbestrol-induced pituitary tumors (DES-T) in rats. Presently, we found that bromocriptine (BROM) treatment increased the levels of GCR in DES-T, demonstrated by steroid binding assays and immunocytochemistry using a monoclonal antibody against the type II GCR. We also found that the high content of nuclear estradiol receptors in the adenomata and the elevated levels of PRL in serum of DES-T were significantly reduced after BROM treatment. In parallel studies, PRL secretion was measured after administration of ether stress. In controls, serum PRL markedly increased after ether and this effect was blunted by prior dexamethasone (DEX) administration, due to the steroid negative feedback on PRL secretion. In animals with DES-T, ether stress had no effect on serum PRL, and the inhibition by DEX was lost unless they received BROM, which restored the negative feedback of DEX on serum PRL. Although increases of PRL titers in pituitary tumors may be due to estrogenic stimulation of lactotroph proliferation and function, coupled to absent dopaminergic inhibition on these cells, other mechanisms are possible. In this respect, inefficient steroid negative feedback on PRL synthesis due to down-regulation of GCR may contribute to hyperprolactinemia. This mechanism is supported from the restoration of GCR and steroid negative feedback on serum PRL by treatment of tumor-bearing rats with BROM.

Background: Previous studies have shown that binding of [3H]cyclic adenosine 3',5'-monophosphate (cAMP) is increased in cytosol of diethylstilbestrol (DES)-induced pituitary tumors. In tumor cells, the cAMP-binding proteins that stimulate cell proliferation have been shown to predominate over those that inhibit it., Purpose: This study was designed to determine the type of regulatory subunit (R) of cAMP-dependent protein kinase (PK) responsible for this binding by determining the type of subunit that is increased in DES-induced pituitary tumors., Methods: Experiments were carried out on three groups of Fischer 344 rats: 1) rats with DES-induced pituitary tumors, 2) ovariectomized rats receiving short-term estrogen treatment with estradiol benzoate (E2) for 4 days, and 3) ovariectomized control rats. We performed immunoprecipitation of RI and RII subunits with polyclonal antibodies in pituitary cytosol (direct method) or after separation of subunits by DEAE-cellulose chromatography (indirect method). The concentration of cAMP was also quantified by radioimmunoassay in pituitaries from the three groups., Results: Direct immunoprecipitation with RI antibody demonstrated a statistically significant increase in [3H]cAMP bound to RI in rats receiving E2 for 4 days over that for control rats and an even more significant increase in rats with DES-induced pituitary tumors. There was little change in the nucleotide [3H]cAMP bound to RII. Immunoprecipitation of the eluted fractions after chromatography demonstrated an RI subunit in peaks 1 and 2, whereas RII was contained almost exclusively in peak 2. After chromatography (indirect method), immunoprecipitation with RI and RII antibody indicated an overall increase in the level of binding to RI protein in tumors. Levels of cAMP in DES-induced pituitary tumors were also high compared with levels in controls or in glands from estrogen-treated rats., Conclusions: In DES-induced pituitary tumors, cAMP may be preferentially bound to one isozyme of PK, which supports current theories that cell proliferation and tumor growth correlate with high expression of the RI subunit., Implications: We plan studies to investigate the effects on tumor growth of the site-selective analogue 8-chloro-cAMP, which binds to RII and causes the elevated levels of the RI subunit of the tumor cells to return to normal levels.

Type I corticosteroid receptors were determined in cytosol from hippocampus (HIPPO) and amygdala (AMYG), using [3H]aldosterone (ALDO), [3H]dexamethasone (DEX) or the mineralocorticoid antagonist [3H]ZK 91587 as ligands. Incubations with the first two compounds also contained the pure glucocorticoid RU 28362 to block type II receptors. Binding of the three ligands was comparable in cytosol from HIPPO and it was slightly higher for [3H]DEX in AMYG. However, after heat-induced receptor transformation, binding to DNA-cellulose was observed for [3H]ALDO-receptor complex obtained from HIPPO or AMYG, whereas it was negligible for [3H]ZK 91587. Receptors charged with [3H]DEX or [3H]ALDO showed similar retention on DNA-cellulose columns in the case of the AMYG, while binding to the polynucleotide was higher for [3H]ALDO in the HIPPO. Finally, only [3H]ALDO was taken up to a significant extent in purified cell nuclei prepared from slices of HIPPO and AMYG previously incubated with the three ligands. It is concluded that binding of a natural agonist steroid may be a prerequisite for type I receptor transformation and translocation from the cytoplasm into the nuclear fraction. DEX binding to type I receptors resembles a partial agonist with antagonist properties, whereas antagonists such as ZK 91587 are bound and retained in cytoplasm, without further translocation.

1. We have used in situ hybridization techniques to determine the mRNA for (Na + K)ATPase in 20 brain regions from control rats and rats treated with high doses of deoxycorticosterone (DOC). 2. DOC-treated rats developed a salt appetite following the second hormone administration on alternate days and were used after the fourth DOC administration. 3. DOC treatment did not change the number of silver grains/cell deposited in cells from Ca1, CA2, CA3, and CA4 hippocampal subfields, dentate gyrus, cerebral cortex, medial preoptic area (POA), substantia nigra, and periventricular gray matter. 4. Nonsignificant reductions were detected in lateral POA, medial and lateral septum, caudate-putamen, and three amygdaloid nuclei (cortical, basolateral, and central) from DOC-treated rats. 5. Significant reductions were obtained, after DOC administration, in arcuate and ventromedial hypothalamic nuclei and medial and lateral amygdala. 6. The results suggested that regulation of the beta-subunit mRNA of (Na + K)-ATPase may be related to the central actions of mineralocorticoids in the control of salt intake.

Binding of cyclic adenosine 3',5'-monophosphate (cAMP) and steroid receptors was studied in cytoplasmic and nuclear fractions of pituitaries from castrated rats, in rats subjected to acute (60 min) or short-term (4 days) estradiol (E2) treatment, and in diethylstilbestrol-induced pituitary tumors (DES-T). E2 receptors were primarily in nuclear extracts in all animals that were given estrogens, whereas cytosolic receptors were low to absent. Contrarily, castrated rats showed high quantities of cytoplasmic receptor but little in nuclear sites. The progestin receptor was induced only in 4-day E2-treated rats and in DES-T. cAMP binding was stimulated in cytosol from 4-day E2-treated rats and in DES-T, but a significant reduction in binding was also noted in nuclear extracts from DES-T. Scatchard analysis for the cytosolic cAMP-binding activity demonstrated a two-component system, and the increased cAMP binding obtained in DES-T seemed to be caused by an increase in the low-affinity, high-capacity binder [regulatory type II (RII) subunit of protein kinase]. Suggestion of the preferential estrogenic induction of RII was also obtained by DEAE-cellulose chromatography, which provided separation of RI and RII subunits. The results suggest that sustained estrogenization leads to induction of cytosolic cAMP-binding protein and increased levels of nuclear E2 receptor. In DES-T, this effect resulted in an inverse subcellular distribution of both binding proteins, which may be related to abnormal growth of the pituitary, as has been postulated for hormone-dependent mammary tumors.

We have determined binding sites for estrogen, progestin, androgen and glucocorticoid in anterior pituitaries from Sprague-Dawley rats, a strain with low estrogen sensitivity, and in diethylstilbestrol-induced pituitary tumors in Fischer 344 rats, a strain with high estrogen sensitivity. Binding sites differ in their quantity and subcellular distribution. Cytosolic sites for [3H]estradiol in normal pituitaries from untreated rats were high prevailing over sites for other hormones, but they were depleted in the tumors due to their retention in nuclei under the influence of estrogen. Unoccupied nuclear sites for estrogen in normal glands also prevailed over sites for other steroids, and were similar to those in tumors. Second, the progestin site labeled with [3H]R 5020 was concentrated 5.7-fold in cytosol and 8.5-fold in nuclei of the tumors over the values found in glands from normal males estrogenized for 3 days. Third, glucocorticoid receptors labeled with [3H]dexamethasone were predominantly cytosolic in normal glands, but very low in cytosol and more evident in nuclear extracts from the tumors, the reverse of the profile found in normal pituitaries. Last, limited and comparable amounts of androgen receptors were measured in the subcellular fractions of both tissues. It is suggested that the subcellular distribution of some steroid receptors may be controlled in part by the cell population of the tissue and its degree of genetic activity.

Free nuclear receptors were measured in anterior pituitaries from ovariectomized-adrenalectomized rats after extraction of purified nuclei with 0.4 M KC1 and incubation of the extract with 2.5 nM (3H)-estradiol (E2) during 1.5 h at 0-4 degrees C. High affinity, low capacity binding was present in untreated rats; receptor concentration doubled after E2 was given for 4 days, whereas acute (60 min) treatment was without effect. Prolonged exposure to diethylstilbestrol (3 months) down-regulated free as well as total nuclear receptors. Tamoxifen increased free nuclear receptors, induced the progestin receptor in cytosol, and inhibited E2-stimulated serum prolactin but not E2 stimulation of free nuclear receptors. Progesterone and testosterone had no effect on basal or E2-stimulated free nuclear sites, whereas dexamethasone reduced the former but not the latter. Furthermore, from 33 to 36% of free nuclear receptors bound to (3H)-E2, were retained in DNA-cellulose columns whether incubation with ligand was performed at low (0-4 degrees C) or high temperature (25 degrees C); DNA-cellulose binding was unchanged after short E2 but increased after 4 days of E2. These data suggest ligand as well as antiestrogen and corticoid regulation of free nuclear receptors for E2 in anterior pituitary. Free nuclear sites may be a fraction of total cell receptors which are in the transformed state and which bind more tightly to nuclei in vivo, as indicated by the results of DNA-cellulose binding in vitro.