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As climate-related impacts threaten marine biodiversity globally, it is important to adjust conservation efforts to mitigate the effects of climate change. Translating scientific knowledge into practical management, however, is often complicated due to resource, economic and policy constraints, generating a knowledge-action gap. To develop potential solutions for marine turtle conservation, we explored the perceptions of key actors across 18 countries in the Mediterranean. These actors evaluated their perceived relative importance of 19 adaptation and mitigation measures that could safeguard marine turtles from climate change. Of importance, despite differences in expertise, experience and focal country, the perceptions of researchers and management practitioners largely converged with respect to prioritizing adaptation and mitigation measures. Climate change was considered to have the greatest impacts on offspring sex ratios and suitable nesting sites. The most viable adaptation/mitigation measures were considered to be reducing other pressures that act in parallel to climate change. Ecological effectiveness represented a key determinant for implementing proposed measures, followed by practical applicability, financial cost, and societal cost. This convergence in opinions across actors likely reflects long-standing initiatives in the Mediterranean region towards supporting knowledge exchange in marine turtle conservation. Our results provide important guidance on how to prioritize measures that incorporate climate change in decision-making processes related to the current and future management and protection of marine turtles at the ocean-basin scale, and could be used to guide decisions in other regions globally. Importantly, this study demonstrates a successful example of how interactive processes can be used to fill the knowledge-action gap between research and management., This work was conducted under FutureMares EU project that received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 869300. The Mediterranean Marine Turtle Working Group was established in 2017 and is continuously supported by MedPAN and the National Marine Park of Zakynthos. The work of AC was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the “First Call for H.F.R.I. Research Projects to support Faculty members and Researchers and the procurement of high-cost research equipment grant” (Project Number: 2340).

Clinical studies indicate that obese individuals have an increased risk of developing co-morbid depressive illness and that these patients have reduced responses to antidepressant therapy, including selective serotonin reuptake inhibitors (SSRIs). Obesity, a condition of chronic mild inflammation including obesity-induced neuroinflammation, is proposed to contribute to decreases in synaptic concentrations of neurotransmitters like serotonin (5HT) by decreasing 5HT synthesis in the dorsal raphe nucleus (DRN) and/or affecting 5HT reuptake in DRN target regions like the hippocampus. In view of these observations, the goal of the current study was to examine inflammatory markers and serotonergic dynamics in co-morbid obesity and depression. Biochemical and behavioral assays revealed that high-fat diet produced an obesity and depressive-like phenotype in one cohort of rats and that these changes were marked by increases in key pro-inflammatory cytokines in the hippocampus. In real time using fast scan cyclic voltammetry (FSCV), we observed no changes in basal levels of hippocampal 5HT; however responses to escitalopram were significantly impaired in the hippocampus of obese rats compared to diet resistant rats and control rats. Further studies revealed that these neurochemical observations could be explained by increases in serotonin transporter (SERT) expression in the hippocampus driven by elevated neuroinflammation. Collectively, these results demonstrate that obesity-induced increases in neuroinflammation adversely affect SERT expression in the hippocampus of obese rats, thereby providing a potential synaptic mechanism for reduced SSRI responsiveness in obese subjects with co-morbid depressive illness.

The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.

Cell polarity, the asymmetric distribution of proteins, organelles, and cytoskeleton, plays an important role in development, homeostasis, and disease. Understanding the mechanisms that govern cell polarity is critical for creating strategies to treat developmental defects, accelerate tissue regeneration, and hinder cancer progression. This review focuses on the role of cell polarity in a number of physiological processes, including asymmetric division, cell migration, immune response mediated by T lymphocytes, and cancer progression and metastasis, and highlights microfabrication techniques to systematically parse the role of microenvironmental cues in the regulation of cell polarity.

C5a is a crucial terminal effector of the C cascade, mostly involved in pain and neuroinflammatory conditions. DF3016A is a novel potent and selective C5a receptor (C5aR) inhibitor that crosses the blood-brain barrier (BBB) and may have pharmacological properties. We have previously demonstrated a protective effect of DF3016A on injured primary cortical neurons by oxygen-glucose deprivation-reoxygenation (OGD/R) model to mimic the neuroinflammatory process. Here, we investigated the molecular pathway and factors involved in the neuroprotection previously reported. Our findings show that DF3016A protects against the neuroinflammatory insult by activating brain-derived neurotrophic factor (BDNF) transcription pathway, which involves methyl CpG-binding protein 2 (MeCP2) and microRNA-132 (miR-132) regulatory factors, both required in nociceptive signaling and neuroinflammation. Further in vivo investigations will confirm the functionality of the DF3016A molecule as a therapeutic resource in neuroinflammation and pain injuries.

The central nervous system (CNS) constitutively expresses complement (C) membrane receptors and complement proteins, including the component C5a. This is a crucial terminal effector of the C cascade, mostly involved in pain and neuroinflammatory conditions. Aberrant activation of C5a protein and its receptor C5aR has been reported to play a critical role in neurodegenerative diseases, with important clinical consequences. Here we have investigated the effects of DF3016A, a novel selective C5aR antagonist, able to penetrate the blood-brain barrier (BBB), on cortical neurons exposed to oxygen-glucose deprivation-reoxygenation (OGD/R), a neuroinflammation-related process. We demonstrated that a mild ischemic insult induces an early upregulation of C5aR associated with the over-production of pro-inflammatory cytokines and the over-expression of the transcriptional regulatory factor miR-181. Furthermore, we report the first experimental evidence of the effect of DF3016A, modulating complement component C5a, on neurons in a model of injury. Interestingly, DF3016A protects neuronal viability by restoring intracellular calcium levels, thus opposing the increase in pro-inflammatory cytokine levels and miR-181 expression. Based on our results, we suggest that DF3016A is a novel C5aR antagonist promoting protective effects against OGD/R-induced damage that could be a new therapeutic approach to controlling CNS neuroinflammatory conditions.

Insulin receptors (IRs) are expressed in discrete neuronal populations in the central nervous system, including the hippocampus. To elucidate the functional role of hippocampal IRs independent of metabolic function, we generated a model of hippocampal-specific insulin resistance using a lentiviral vector expressing an IR antisense sequence (LV-IRAS). LV-IRAS effectively downregulates IR expression in the rat hippocampus without affecting body weight, adiposity, or peripheral glucose homeostasis. Nevertheless, hippocampal neuroplasticity was impaired in LV-IRAS–treated rats. High-frequency stimulation, which evoked robust long-term potentiation (LTP) in brain slices from LV control rats, failed to evoke LTP in LV-IRAS–treated rats. GluN2B subunit levels, as well as the basal level of phosphorylation of GluA1, were reduced in the hippocampus of LV-IRAS rats. Moreover, these deficits in synaptic transmission were associated with impairments in spatial learning. We suggest that alterations in the expression and phosphorylation of glutamate receptor subunits underlie the alterations in LTP and that these changes are responsible for the impairment in hippocampal-dependent learning. Importantly, these learning deficits are strikingly similar to the impairments in complex task performance observed in patients with diabetes, which strengthens the hypothesis that hippocampal insulin resistance is a key mediator of cognitive deficits independent of glycemic control.

The cannabinoid CB1 receptor is expressed throughout the central nervous system where it functions to regulate neurotransmitter release and synaptic plasticity. While the CB1 receptor has been identified as a target for both natural and synthetic cannabinoids, the specific downstream signaling pathways activated by these various ligands have not been fully described. In this study, we developed a real-time membrane potential fluorescent assay for cannabinoids using pituitary AtT20 cells that endogenously express G protein-gated inward rectifier K+ (GIRK) channels and were stably transfected with the CB1 receptor using a recombinant lentivirus. In whole-cell patch clamp experiments application of the cannabinoid agonist WIN 55,212-2 to AtT20 cells expressing the CB1 receptor (AtT20/CB1) activated GIRK currents that were blocked by BaCl2. WIN 55,212-2 activation of the GIRK channels was associated with a time- and concentration-dependent (EC50 = 309 nM) hyperpolarization of the membrane potential in the AtT20/CB1 cells when monitored using a fluorescent membrane potential-sensitive dye. The WIN 55,212-2-induced fluorescent signal was inhibited by pretreatment of the cells with either the GIRK channel blocker tertiapin-Q or the CB1 receptor antagonist SR141716. The cannabinoids displayed a response of WIN 55,212-2 ≈ anandamide (AEA) > CP 55,940 > Δ9-tetrahydrocannabinol (THC) when maximal concentrations of the four ligands were tested in the assay. Thus, the AtT20/CB1 cell fluorescent assay will provide a straightforward and efficient methodology for examining cannabinoid-stimulated Gi signaling.

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.

Protein succination by fumarate increases in the adipose tissue of diabetic mice and in adipocytes matured in high glucose as a result of glucotoxicity-driven mitochondrial stress. The endoplasmic reticulum (ER) oxidoreductase protein disulfide isomerase (PDI) is succinated in adipocytes that are matured in high glucose, and in this study we investigated whether succination would alter PDI oxidoreductase activity, directly linking mitochondrial stress and ER stress.Protein succination and the ER stress marker C/EBP homologous protein (CHOP) were diminished after pharmaceutical targeting of mitochondrial stress with the chemical uncoupler niclosamide in adipocytes matured in high-glucose concentrations. PDI was succinated by fumarate on both CXXC-containing active sites, contributing to reduced enzymatic activity. Succinated PDI decreased reductase activity in adipocytes matured in high glucose, and in db/db epididymal adipose tissue, in association with increased levels of CHOP. PDI succination was increased in fumarase knockdown adipocytes, leading to reduced PDI oxidoreductase activity, increased CHOP levels, and pro-inflammatory cytokine secretion, confirming the specific role of elevated fumarate levels in contributing to ER stress. In addition, PDI succination and ER stress were decreased, and PDI reductase activity was restored when exposure to chronic high glucose was limited, highlighting the importance of calorie restriction in the improvement of adipocyte metabolic function.These experiments identify PDI succination as a novel biochemical mechanism linking altered mitochondrial metabolism to ER stress in the adipocyte during diabetes.The current study demonstrates that early biochemical changes in mitochondrial metabolism have important implications for the development of adipocyte stress. Antioxid. Redox Signal. 27, 1281-1296.

Enzymes of central carbon metabolism are essential mediators of Mycobacterium tuberculosis (Mtb) physiology and pathogenicity, but are often perceived to lack sufficient species selectivity to be pursued as potential drug targets. Fumarase (Fum) is an enzyme of the canonical tricarboxylic acid cycle and is dispensable in many organisms. Transposon mutagenesis studies in Mtb, however, indicate that Fum is required for optimal growth. Here, we report the generation and characterization of a genetically engineered Mtb strain in which Fum expression is conditionally regulated. This revealed that Fum deficiency is bactericidal in vitro and during both the acute and chronic phases of mouse infection. This essentiality is linked to marked accumulations of fumarate resulting in protein and metabolite succination, a covalent modification of cysteine thiol residues. These results identify Mtb Fum as a potentially species-specific drug target whose inactivation may kill Mtb through a covalently irreversible form of metabolic toxicity.

Stress is a common environmental factor associated with depressive illness and the amygdala is thought to be integral for this association. For example, repeated stress impairs amygdalar neuroplasticity in rodents and these defects parallel amygdalar deficits in depressive illness patients. Because the excitatory neurotransmitter glutamate is important in neuroplasticity, we hypothesized that alterations in amygdalar glutamatergic systems may serve as key players in depressive illness. Moreover, restoration of amygdalar glutamatergic systems may serve as important therapeutic targets in the successful management of multiple stress-related mood disorders. To address these hypotheses, we measured glutamate efflux in the basolateral and central amygdalar complexes via in vivo microdialysis, as well as the expression of synaptic proteins that regulate vesicular glutamate packaging and release, in rats subjected to repeated stress and treated daily with saline or the antidepressant tianeptine. Glutamate efflux was significantly reduced in the central amygdalar complex of animals subjected to repeated stress. In addition, repeated stress nearly eliminated amygdalar vGLUT2 expression, thereby proving a potential mechanism through which repeated stress impairs amygdalar glutamate neurochemistry. These stress-induced changes in glutamate efflux and vGLUT2 expression were inhibited by daily tianeptine administration. Moreover, tianeptine administration increased the vesicular localization of SNAP-25, which could account for the ability of tianeptine to modify glutamatergic tone in non-stressed control rats. Collectively, these results demonstrate that repeated stress differentially affects amygdalar glutamate systems and further supports our previous studies indicating that tianeptine's antidepressant efficacy may involve targeting amygdalar glutatamatergic systems.

Ectonucleotidases and the nucleotide metabolism have been implicated as important regulators in diabetes disease. We evaluated the ectonucleotidase activities and biochemical parameters in blood serum of streptozotocin (STZ)-induced diabetic rats submitted a physical training protocol. We observed a raise in ATP, ADP, AMP and p-Nph-5'-TMP hydrolysis rate and in the levels of cholesterol and triglycerides in rat blood serum, after 30 days of diabetes induction. However, in serum of rats submitted a physical training protocol by forced swimming, both the nucleotide hydrolysis rate and the lipids levels returned to the control values. Considering that diabetes leads to multiple pathophysiological alterations, the modulations observed in ectonucleotidase activities may be part of the events involved in these alterations. Then the physical training is a very important way to control the vascular alterations developed in diabetes.

Epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese and there is a growing appreciation that obesity affects the functional integrity of the central nervous system (CNS). We recently developed a lentivirus (LV) vector that produces an insulin receptor (IR) antisense RNA sequence (IRAS) that when injected into the hypothalamus selectively decreases IR signaling in hypothalamus, resulting in increased body weight, peripheral adiposity and plasma leptin levels. To test the hypothesis that this obesity/hyperleptinemic phenotype would impair hippocampal synaptic transmission, we examined short term potentiation (STP) and long term potentiation (LTP) in the hippocampus of rats that received the LV-IRAS construct or the LV-Control construct in the hypothalamus (hypo-IRAS and hypo-Con, respectively). Stimulation of the Schaffer collaterals elicits STP that develops into LTP in the CA1 region of hypo-Con rats; conversely, hypo-IRAS rats exhibit STP that fails to develop into LTP. To more closely examine the potential role of hyperleptinemia in these electrophysiological deficits, hypo-IRAS were subjected to mild food restriction paradigms that would either: 1) prevent the development of the obesity phenotype; or 2) reverse an established obesity phenotype in hypo-IRAS rats. Both of these paradigms restored LTP in the CA1 region and reversed the decreases in the phosphorylated/total ratio of GluA1 Ser845 AMPA receptor subunit expression observed in the hippocampus of hypo-IRAS rats. Collectively, these data support the hypothesis that obesity impairs hippocampal synaptic transmission and support the hypothesis that these deficits are mediated through the impairment of hippocampal leptin activity.

An increased activity of the hypothalamo-pituitary-adrenal axis resulting in exaggerated glucocorticoid secretion has been repeatedly described in patients with diabetes mellitus and in animal models of this disease. However, it has been pointed out that experimental diabetes is accompanied by a decreased glucocorticoid response to ACTH stimulation. Because previous studies from our laboratory demonstrate the involvement of nitric oxide (NO) in the modulation of corticosterone production, present investigations were designed to evaluate 1) the impact of streptozotocin (STZ)-induced diabetes on the adrenocortical nitrergic system and 2) the role of NO in the modulation of adrenal steroidogenesis in STZ-diabetic rats. Four weeks after STZ injection, increased activity and expression levels of proteins involved in l-arginine transport and in NO synthesis were detected, and increased levels of thiobarbituric acid reactive species, carbonyl adducts, and nitrotyrosine-modified proteins were measured in the adrenocortical tissue of hyperglycemic rats. An impaired corticosterone response to ACTH was evident both in vivo and in adrenocortical cells isolated from STZ-treated animals. Inhibition of NO synthase activity resulted in higher corticosterone generation in adrenal tissue from STZ-treated rats. Moreover, a stronger inhibition of steroid output from adrenal cells by a NO donor was observed in adrenocortical Y1 cells previously subjected to high glucose (30 mm) treatment. In summary, results presented herein indicate an inhibitory effect of endogenously generated NO on steroid production, probably potentiated by hyperglycemia-induced oxidative stress, in the adrenal cortex of STZ-treated rats.

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 pre-treatment 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.

Central nervous system (CNS) complications resulting from diabetes is a problem that is gaining more acceptance and attention. Recent evidence suggests morphological, electrophysiological and cognitive changes, often observed in the hippocampus, in diabetic individuals. Many of the CNS changes observed in diabetic patients and animal models of diabetes are reminiscent of the changes seen in normal aging. The central commonalities between diabetes-induced and age-related CNS changes have led to the theory of advanced brain aging in diabetic patients. This review summarizes the findings of the literature as they relate to the relationship between diabetes and dementia and discusses some of the potential contributors to diabetes-induced CNS impairments.

Recent studies have demonstrated that adult humans have substantial amounts of functioning brown adipose tissue (BAT). Since BAT has been implicated as an anti-obese and anti-diabetic tissue, it is important to understand the signaling molecules that regulate BAT function. There has been a link between insulin signaling and BAT metabolism as deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function. Tribbles 3 (TRB3) is a pseudo kinase that has been shown to regulate metabolism and insulin signaling in multiple tissues but the role of TRB3 in BAT has not been studied. In this study, we found that TRB3 expression was present in BAT and overexpression of TRB3 in brown preadipocytes impaired differentiation and decreased expression of BAT markers. Furthermore, TRB3 overexpression resulted in significantly lower oxygen consumption rates for basal and proton leakage, indicating decreased BAT activity. Based on previous studies showing that deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function, we assessed insulin signaling in brown preadipocytes and BAT in vivo. Overexpression of TRB3 in cells impaired insulin-stimulated IRS1 and Akt phosphorylation, whereas TRB3KO mice displayed improved IRS1 and Akt phosphorylation. Finally, deletion of IRS1 abolished the function of TRB3 to regulate BAT differentiation and metabolism. These data demonstrate that TRB3 inhibits insulin signaling in BAT, resulting in impaired differentiation and function.

We thank Ms. Jennifer Bethard, Medical University of South Carolina for the mass spectrometric analysis of the 2D gel spots. We would like to thank Professor John Baynes, USC for helpful discussion. We thank Dr. Boris Kantor, USC Viral Vector Core for production of the lentiviral vectors. This work was supported by grants from the National Institutes of Health (R03 HD077187, R01 NS092938, R37 DK19971 and P20 GM109091), the American Diabetes Association (1-11-JF-13), and a University of South Carolina Research Foundation ASPIRE-I award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-Translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltagedependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys77 and Cys48 were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.

Background: In the rat hippocampus, the predominate glutamate transporters are GLT-1 and its recently identified isoform, GLT-1b. Chronic restraint stress increases GLT-1b expression throughout the hippocampus while more selectively increasing GLT-1 expression in the CA3 region. These studies suggest that GLT-1b expression is regulated by stress levels of glucocorticoids (GCs) and GLT-1 expression is regulated by stress-induced increases in extracellular glutamate levels in the CA3 region. Methods: In order to differentiate between the actions of GCs and glutamate, we examined GLT-1 isoform expression in adrenalectomized (ADX) rats and rats exposed to stress levels of GCs. Results: ADX rats revealed no significant differences in GLT-1b mRNA or protein levels compared to sham-operated controls or ADX rats given GC replacement. However, rats exposed to stress levels of GCs exhibited increases in GLT-1b protein expression in the CA3 region and the dentate gyrus. GLT-1 mRNA expression was increased by ADX, increases that were inhibited by GC replacement. Similarly, stress levels of GCs increased GLT-1 protein expression throughout the hippocampus. Conclusions: Taken together, these data indicate that GLT-1b protein expression is regulated by stress levels of GCs while the regulation of GLT-1 mRNA and protein expression provides another example of the biphasic actions of GCs in the central nervous system.

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.

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.

Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate 2SC [S-(2-succino)cysteine]. We demonstrate that both α- and β-tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db / db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound DMF (dimethylfumarate, 500 μM) inhibited polymerization up to 35% and 59% respectively. Using MS we identified Cys347α, Cys376α, Cys12β and Cys303β as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteine residues increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose compared with normal glucose also had reduced reactivity with the anti-α-tubulin antibody; suggesting that succination may interfere with tubulin–protein interactions. DMF reacted rapidly with 11 of the 20 cysteine residues in the αβ-tubulin dimer, decreased the number of free thiols and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggest that inhibition of tubulin polymerization is an important undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics. Abbreviations: C2SC, cysteine succination by fumarate; CID, collision-induced dissociation; CTT, C-terminal tail of tubulin; DMF, dimethylfumarate; DMEM, Dulbecco’s modified Eagle’s medium; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); DTPA, diethylenetriaminepenta-acetic acid; GI, gastrointestinal; 4-HNE, 4-hydroxynonenal; MAP, microtubule-associated protein; NEM, N-ethylmaleimide; PE, pyridylethylation; PTM, post-translational modification; RIPA, radioimmunoprecipitation assay; 2SC, S-2-(succino)cysteine; TFA, trifluoroacetic acid; TOF, time-of-flight

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.

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.

Chronic exposure of F344 rats to diethylstilbestrol (DES) induces pituitary tumors (DES-T) composed of proliferating lactotrophs. Presently, we studied the effect of progestins on parameters related to tumor growth and function, due to previous evidences of progesterone antagonism of pituitary tumorigenesis acting at pituitary and hypothalamic levels [Piroli, G., Grillo, C., Ferrini, M., Lux-Lantos, V. and De Nicola, A. F., Antagonism by progesterone of diethylstilbestrol-induced pituitary tumorigenesis in Fischer 344 rats: Effects on sex steroid receptors and tyrosine hydroxylase mRNA, Neuroendocrinology, 1996, 63, 530-539]. In search of a quantitatively more important effect, animals bearing DES-T were treated with synthetic progestins. Competition assays using DES-T as source of progestin receptors indicated that levonorgestrel (LNG), gestodene and R5020 showed higher affinities (IC50 1-2 nM) than progesterone, norethisterone and medroxyprogesterone (IC50 10-25 nM). Treatment with LNG reduced DES-T weight by 45%, and serum PRL by one half. Small (monomeric) and big (polymeric) PRL increased 5- and 2.5-fold, respectively, in DES-T in comparison with pituitaries of ovariectomized (OVX) rats. However, LNG produced no changes indicating that synthesis and storage of PRL was conserved in rats receiving both hormonal treatments. DES induced a 15-fold increase in cell proliferation, measured as bromodeoxyuridine incorporation into cell nuclei, in comparison to OVX rats, while LNG treatment of DES-T bearing rats reduced this index by 72%. Electron microscopic images showed that LNG markedly reduced hypertrophy and hyperplasia of lactotropes, increasing the proportions of degenerating cells and cells of high electronic density with alterations of cytoplasmic organelles. However, histopathological signs of apoptosis were absent. Therefore, reduced cell proliferation and non-apoptotic cell death are part of the mechanisms employed by progestins to antagonize tumorigenesis at the pituitary level. The results may open a new therapeutic strategy for treatment of PRL secreting adenoma 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.

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 α 3 and β 1 subunits of the (Na,K)-ATPase because this enzyme could by a mediator of MC action in target cells. We employed [ 35 S]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 α 3 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 β 1 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 α 3 and β 1 subunit mRNA in the hippocampus, gyrus dentatus and periventricular gray substance, whereas in other regions the stimulatory effect was exclusive of the α 3 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.

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.

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.

Ongoing epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese. There is a growing appreciation that the complications of obesity extend to the central nervous system (CNS) and may result in increased risk for neurological co-morbidities like depressive illness. One potential mechanistic mediator linking obesity and depressive illness is the adipocyte derived hormone leptin. We previously demonstrated that lentivirus-mediated downregulation of hypothalamic insulin receptors increases body weight, adiposity and plasma leptin levels, which is consistent with features of the metabolic syndrome. Using this novel model of obesity, we examined performance in the forced swim test (FST), the sucrose preference test and the elevated plus maze (EPM), approaches that are often used as measures of depressive-like and anxiety-like behaviors, in rats that received third ventricular injections of either an insulin receptor antisense lentivirus (hypo-IRAS) or a control lentivirus (hypo-Con). Hypo-IRAS rats exhibited significant increases in immobility time and corresponding decreases in active behaviors in the FST and exhibited anhedonia as measured by decreased sucrose intake compared to hypo-Con rats. Hypo-IRAS rats also exhibited increases in anxiety-like behaviors in the EPM. Plasma, hippocampal and amygdalar brain-derived neurotrophic factor (BDNF) levels were reduced in hypo-IRAS rats, suggesting that the obesity/hyperleptinemic phenotype may elicit this behavioral phenotype through modulation of neurotrophic factor expression. Collectively, these data support the hypothesis for an increased risk for mood disorders in obesity, which may be related to decreased expression of hippocampal and amygdalar BDNF.

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.

Using both IN VITRO and IN VIVO approaches, we studied the antagonism exerted by the synthetic progestin levonorgestrel on estrogen-induced prolactinomas, considering that levonorgestrel shows partial androgenic properties and that androgens inhibit estrogen-induced prolactin synthesis and secretion. In the tumors, binding of estrogens to their receptors was competed neither by progesterone receptor ligands nor by androgen receptor ligands, ruling out direct inhibitory effects of these drugs on tumor development. Progestin binding was competed by the progesterone receptor agonists progesterone and levonorgestrel, by the antagonist mifepristone, and also by the androgen dihydrotestosterone, whereas the androgen receptor antagonist hydroxyflutamide was a weak competitor. In addition, both progesterone receptor and androgen receptor ligands competed for binding to androgen receptors. In primary cultures of pituitary tumors, levonorgestrel decreased prolactin secretion, an effect that was blocked by mifepristone but not by hydroxyflutamide. IN VIVO results indicated that levonorgestrel inhibition of both estrogen-induced pituitary weight increment and hyperprolactinemia was reduced by mifepristone, whereas flutamide was unable to block levonorgestrel effects. Our results suggest that even when an interaction of levonorgestrel with androgen receptors in the tumors is possible, the antagonistic effects of levonorgestrel on tumor development and functionality are mediated by progesterone receptors.

Development of cognitive-enhancing drugs that delay or halt mild cognitive impairment progression to Alzheimer's disease would be of great benefit.The aim of this study was to examine the ability of (S)-2,3-dihydro-[3,4]-cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S 18986), a positive allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, to improve behavioral performance and alleviate age-related deficits in oxidative stress status in the prelimbic cortex and hippocampus.Daily administration of S 18986 (0.1, 0.3, and 1.0 mg/kg) or vehicle was given to separate groups of male rats starting at 12 months of age. Additionally, daily vehicle administration was given to a group of rats starting at 3 months of age. Four months after initiation of drug administration, rats were trained and tested in an operant-delayed alternation task and a reinforcer devaluation task. Upon completion of testing, oxidative stress status was assessed in the prelimbic cortex and hippocampus.S 18986 dose-dependently altered responses in the reinforcer devaluation task such that aged rats came to resemble young rats. There were no age or drug effects in the operant-delayed alternation task. Levels of the lipid peroxidation product 4-hydroxy-nonenal (HNE) were increased, and Cu/Zn-superoxide dismutase (SOD) levels were decreased in prelimbic cortex in aged rats, changes that were reversed by S 18986. Similarly, age-related increases in hippocampal HNE levels were prevented by S 18986.Positive modulation of AMPA receptor activity may be a therapeutic approach to halt or slow progression of mild cognitive impairment via improvement in oxidative stress status in the hippocampus and prelimbic cortex.

Unlike responses to acute stressful events that are protective and adaptive in nature, chronic stress elicits neurochemical, neuroanatomical and cellular changes that may have deleterious consequences upon higher brain functioning. For example, while exposure to acute stress facilitates memory formation and consolidation, chronic stress or chronic exposure to stress levels of glucocorticoids impairs cognitive performance. Chronic stress or glucocorticoid exposure, as well as impairments in hypothalamic-pituitary-adrenal (HPA) axis function are proposed to participate in the etiology and progression of neurological disorders such as depressive illness, anxiety disorders and post-traumatic stress disorder (PTSD). HPA axis dysfunction, impaired stress responses and elevated basal levels of glucocorticoids are also hallmark features of experimental models of type 1 and type 2 diabetes, as well as diabetic subjects in poor glycemic control. Such results suggest that stress and glucocorticoids contribute to the neurological complications observed in diabetes patients. Interestingly, many of the hyperglycemia mediated changes in the brain are similar to those observed in depressive illness patients and in experimental models of chronic stress. Such results suggest that common mechanisms may be involved in the development of the neurological complications associated with Anxiety, Depressive illness and Diabetes: the As and Ds of stress. The aim of the current review will be to discuss the mechanisms through which limbic structures such as the hippocampus and amygdala respond and adapt to the deleterious consequences of chronic stress and hyperglycemia.

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.

Depressive illness is associated with changes in amygdalar volume, and stressful life events are known to precipitate depressive episodes in this patient population. Stress affects amygdalar synaptic plasticity and several neurotransmitter systems have been implicated in stress-mediated changes in the brain, including the glutamatergic system. However, the role of the glutamatergic system in stress-mediated plasticity in the amygdala remains to be determined. Accordingly the current study examined the stress modulation of extracellular glutamate levels in the basolateral nucleus (BLA) and the central nucleus (CeA) of the amygdala by in vivo microdialysis. Acute stress increased extracellular glutamate levels in the BLA and CeA, although the dynamics of these stress-mediated changes were dramatically different in these amygdalar nuclei. Tetrodotoxin administration reduced basal, and completely eliminated stress-mediated increases in glutamate efflux in the amygdala, demonstrating that stress effects are dependent on local axonal depolarization. Moreover, stress-mediated increases in glutamate efflux in the BLA were inhibited by the antidepressant tianeptine but not by the selective serotonin-reuptake inhibitor fluoxetine. Collectively, these data demonstrate that stress-induced modulation of glutamate neurochemistry reflects a fundamental pathological change that may contribute to the aetiology and progression of depressive illness, and suggest that some antidepressants such as tianeptine may elicit their clinical effects by modulation of glutamatergic neurotransmission.

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.

Background: Exposure to stress levels of glucocorticoids produces physiological responses that are characteristic of type 2 diabetes, such as peripheral insulin resistance and impairment in insulin-stimulated trafficking of glucose transporter 4 (GLUT4) in muscle and fat. In the central nervous system, stress produces neuroanatomical and neurochemical changes in the hippocampus that are associated with cognitive impairments. Methods: In view of these observations, the current studies examined the effects of short-term (1 week) exposure of stress levels of glucocorticoids upon insulin receptor (IR) expression and signaling, including GLUT4 translocation, in the rat hippocampus. Results: One week of corticosterone (CORT) treatment produced insulin resistance in response to peripheral glucose challenge. In the hippocampus, IR expression was unchanged in CORT-treated rats as compared with vehicle-treated rats. However, insulin-stimulated phosphorylation of the IR, total Akt levels and total GLUT4 levels were reduced in CORT-treated rats when compared to controls. In addition, insulin-stimulated translocation of hippocampal GLUT4 to the plasma membrane was completely abolished in CORT-treated rats. Conclusions: These results demonstrate that in addition to eliciting peripheral insulin resistance, short-term CORT administration impairs insulin signaling in the rat hippocampus, effects that may contribute to the deleterious consequences of hypercortisolemic/hyperglycemic states observed in type 2 diabetes.

Chronic restraint stress affects hippocampal and amygdalar synaptic plasticity as determined by electrophysiological, morphological and behavioral measures, changes that are inhibited by some but not all antidepressants. The efficacy of some classes of antidepressants is proposed to involve increased phosphorylation of cAMP response element binding protein (CREB), leading to increased expression of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF). Conversely, some studies suggest that acute and chronic stress downregulate BDNF expression and activity. Accordingly, the aim of the current study was to examine total and phosphorylated CREB (pCREB), as well as BDNF mRNA and protein levels in the hippocampus and amygdala of rats subjected to chronic restraint stress in the presence and absence of the antidepressant tianeptine. In the hippocampus, chronic restraint stress increased pCREB levels without affecting BDNF mRNA or protein expression. Tianeptine administration had no effect upon these measures in the hippocampus. In the amygdala, BDNF mRNA expression was not modulated in chronic restraint stress rats given saline in spite of increased pCREB levels. Conversely, BDNF mRNA levels were increased in the amygdala of chronic restraint stress/tianeptine rats in the absence of changes in pCREB levels when compared to non-stressed controls. Amygdalar BDNF protein increased while pCREB levels decreased in tianeptine-treated rats irrespective of stress conditions. Collectively, these results demonstrate that tianeptine concomitantly decreases pCREB while increasing BDNF expression in the rat amygdala, increases in neurotrophic factor expression that may participate in the enhancement of amygdalar synaptic plasticity mediated by tianeptine.

The genetically obese Zucker rat is a widely investigated model of pathological changes associated with type 2 diabetes. To assess cognitive function, obese and lean Zucker rats were tested on a variable-interval delayed alternation test of learning and memory. There were no group differences in learning the alternation rule or at short intervals, but obese rats were impaired at longer intervals where performance is hippocampus dependent. Plasma membrane association of the insulin sensitive glucose transporter, GLUT4, was reduced in the hippocampus of obese rats in the absence of changes in total GLUT4 and insulin receptor expression. These results parallel those of human studies in pointing to the susceptibility of the hippocampus and related structures to the adverse environment of diabetes mellitus.

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.

Adrenomedullin (AM), inducing a potent and powerful hypotensive activity caused by dilatation of resistance vessels, has elicited interest for its cardiovascular actions. AM is secreted from various cell type, including vascular endothelial and smooth muscle cell. AM plasma levels are increased in various cardiovascular diseases as heart failure and hypertension and may be involved in pathophysiological changes in cardiovascular diseases.

We have previously described a sexual dimorphism in oestrogen-induced anterior pituitary tumorigenesis in Fischer 344 rats, with female tumours averaging twice the size of those of males. Neonatal androgenization of female Fischer 344 rats with 100 µg of testosterone propionate reverted that effect, causing a 'male-like' phenotype. The peptides galanin and vasoactive intestinal peptide (VIP) are possible mediators of oestrogen effects on the anterior pituitary, including hyperprolactinemia and lactotroph proliferation. To further extend our previous findings, we investigated the expression of galanin and VIP in the anterior pituitary of control and oestrogenized male, female and neonatally androgenized female Fischer 344 rats. At 3 months of age, rats were deprived of their gonads and divided into control and diethylstilbestrol (DES)-treated groups. In the anterior pituitary of control rats, galanin and VIP immunoreactive cells were absent. However, in DES-treated rats, pituitaries from normal ovariectomized females showed higher number of galanin and VIP positive cells than pituitaries from neonatally androgenized ovariectomized females and gonadectomized males. This pattern correlated with changes in anterior pituitary weight and serum prolactin. Our study suggests that sexual differences in oestrogen-induced pituitary tumorigenesis could be due to the differential expression of galanin and VIP. Furthermore, our data support the fact that neonatal exposure to androgens, as in normal males and androgenized females, may condition the response of the pituitary gland to oestrogens in adult life. Fil: Piroli, Gerardo Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina Fil: Pietranera, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina Fil: Grillo, Claudia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina Fil: de Nicola, Alejandro Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina

Previous studies show that stress cross-sensitizes with or alters amphetamine (AMPH) effects in male rats; however, few studies include females. We investigated combining daily restraint stress (21 days for 6 h/day) with chronic AMPH (10 injections every other day) on locomotor activity, exploratory activity in an open field and object recognition, a memory task, in female rats. A synaptic protein, synaptophysin, was also quantified by radioimmunocytochemistry (RICC) in brain to determine possible mechanisms for behavioral changes. Beginning at 5 days after cessation of treatments, AMPH increased locomotion, modified exploration, impaired object recognition, and increased serum corticosterone (CORT) levels. Stress did not alter these parameters but blocked AMPH effects on exploration and object recognition, potentiated AMPH-dependent locomotor effects, and did not alter increased CORT levels. AMPH treatment decreased synatophysin expression in the hippocampus. In the caudate nucleus, the AMPH group showed increased synaptophysin expression which was reversed by stress. These results in females corroborate previously shown cross-sensitizations between stress and AMPH for locomotion in males and demonstrate that chronic stress counteracts AMPH-dependent impairments in recognition memory. Stress may counteract AMPH effects on the memory task by blocking both the induction of AMPH anxiety-like effects and neuroplastic changes in the caudate nucleus of female rats.

Disease states such as diabetes mellitus are known to impair hippocampal glucoregulatory activities, which may contribute to cognitive deficits observed in diabetic subjects. Stress or exposure to stress levels of glucocorticoids (GCs) are also intimately involved in hippocampal glucoregulatory activities and the actions of GCs are often most evident in hyperglycemic states. Glucose transporter (GLUT) expression, activity and translocation represent components of the glucoregulatory activities of the hippocampus that may be disrupted by diabetes and stress. Accordingly, the current study examined the effects of stress, streptozotocin (STZ)-induced diabetes and the combined actions of stress and hyperglycemia upon GLUT8 mRNA expression, protein levels and intracellular trafficking in the rat hippocampus. Short-term stress in euglycemic rats had no effect upon GLUT8 mRNA, while restraint stress normalized diabetes mediated increases in GLUT8 mRNA expression in STZ treated rats. Radioimmunocytochemical analysis revealed that total GLUT8 protein levels were not altered by diabetes, short-term stress or the combined actions of hyperglycemia and stress. However, subcellular compartmentalization of GLUT8 was modulated by stress in that hippocampal GLUT8 protein levels were increased in high-density microsomal (HDM) fractions isolated from rats subjected to stress. In contrast, STZ-diabetes decreased GLUT8 protein levels in the HDM fraction, an effect that was potentiated by stress. Collectively, these results demonstrate that the actions of GCs may be dramatically different in euglycemic and hyperglycemic/insulinopenic states, suggesting that stress may increase hippocampal neuronal responsiveness under normal physiological conditions while increasing hippocampal neuronal vulnerability in pathophysiological settings such as in type 1 diabetes.

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.

The expression and localization of glucose transporter isoforms play essential roles in the glucoregulatory activities of the hippocampus and ultimately contribute to cognitive status in physiological and pathophysiological settings. The recently identified glucose transporter GLUT8 is uniquely expressed in neuronal cell bodies in the rat hippocampus and therefore may contribute to hippocampal glucoregulatory activities. We show here that GLUT8 has a novel intracellular distribution in hippocampal neurons and is translocated to intracellular membranes following glucose challenge. Immunoblot analysis revealed that GLUT8 is expressed in high-density microsomes (HDM), suggesting that GLUT8 is associated with intracellular organelles under basal conditions. Immunogold electron microscopic analysis confirmed this observation, in that GLUT8 immunogold particles were associated with the rough endoplasmic reticulum (ER) and cytoplasm. Peripheral glucose administration produced a rapid twofold increase in GLUT8 levels in the HDM fraction while decreasing GLUT8 levels in low-density microsomes. Similarly, peripheral glucose administration significantly increased GLUT8 association with the rough ER in the hippocampus. Conversely, under hyperglycemic/insulinopenic conditions, namely, in streptozotocin (STZ) diabetes, hippocampal GLUT8 protein levels were decreased in the HDM fraction. These results demonstrate that GLUT8 undergoes rapid translocation to the rough ER in the rat hippocampus following peripheral glucose administration, trafficking that is impaired in STZ diabetes, suggesting that insulin serves as a stimulus for GLUT8 translocation in hippocampal neurons. Because glucose is liberated from oligosaccharides during N-linked glycosylation events in the rough ER, we propose that GLUT8 may serve to transport glucose out of the rough ER into the cytosol and in this manner contribute to glucose homeostasis in hippocampal neurons.

The effects of dehydroepiandrosterone (DHEA) on galanin (GAL) and prolactin (PRL) mRNA expression in the anterior pituitary of Fischer 344 rats were studied, taking in consideration that: (1) DHEA is an androgen with estrogenic activity on pituitary lactotrophs; (2) estrogens induce prolactinomas in Fischer 344 rats; and (3) GAL has been considered the main mediator of estrogen-induced lactotroph proliferation.Female rats were ovariectomized and used as controls or treated during 2 weeks with DHEA (500 mg/kg/day or 5 mg/kg/day or 50 mg/kg/day) or estradiol (E2, 50 microg/kg/day), as a positive control for pituitary growth and GAL induction. GAL and PRL mRNA expression were studied by in situ hybridization.Both DHEA and E2 induced PRL mRNA synthesis. However, DHEA neither produced pituitary enlargement nor GAL induction, in contrast to E2.Our results shows that GAL is not involved in the estrogenic activity of DHEA on pituitary lactotrophs, and suggest that DHEA effects are exerted directly on the PRL gene or through another mechanism(s) not related to GAL.