Testosterone Reduces Myelin Abnormalities in the Wobbler Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron degenerative disease that is associated with demyelination. The Wobbler (WR) mouse exhibits motoneuron degeneration, gliosis and myelin deterioration in the cervical spinal cord. Since male WRs display low testosterone (T) levels in the nervous system, we investigated if T modified myelin-relative parameters in WRs in the absence or presence of the aromatase inhibitor, anastrozole (A). We studied myelin by using luxol-fast-blue (LFB) staining, semithin sections, electron microscopy and myelin protein expression, density of IBA1+ microglia and mRNA expression of inflammatory factors, and the glutamatergic parameters glutamine synthetase (GS) and the transporter GLT1. Controls and WR + T showed higher LFB, MBP and PLP staining, lower g-ratios and compact myelin than WRs and WR + T + A, and groups showing the rupture of myelin lamellae. WRs showed increased IBA1+ cells and mRNA for CD11b and inflammatory factors (IL-18, TLR4, TNFαR1 and P2Y12R) vs. controls or WR + T. IBA1+ cells, and CD11b were not reduced in WR + T + A, but inflammatory factors' mRNA remained low. A reduction of GS+ cells and GLT-1 immunoreactivity was observed in WRs and WR + T + A vs. controls and WR + T. Clinically, WR + T but not WR + T + A showed enhanced muscle mass, grip strength and reduced paw abnormalities. Therefore, T effects involve myelin protection, a finding of potential clinical translation.

Effects of metformin on behavioral alterations produced by chronic sucrose consumption in male rats.

Excessive consumption of sugary drinks negatively impacts the developing brain, producing long-lasting behavioral and metabolic disorders. Here, we study whether treatment with the antihyperglycemic agent metformin prevents some of the anxiety and memory alterations produced by chronic sucrose consumption. Male Sprague-Dawley rats had unrestricted access to water (control group) and a bottle containing a 10% sucrose solution (sucrose group, SUC) for 35 days. In parallel, a group of animals from SUC received metformin (25 mg/kg or 50 mg/kg, orally; MET 25 and MET 50 groups, respectively). After 2 weeks of metformin treatment, the animals weighed less than controls. SUC and MET 50 groups compensated for the caloric intake from the sugary solution by consuming less chow. In contrast, total energy intake in MET 25 was higher than the rest of the groups, but they still weighed less than control and SUC groups, suggesting that at this concentration, metformin delays body growth. The animals were then tested for the open field (OF), elevated plus maze (EPM) and novel object location (NOL) tests. In the OF, SUC animals spent more time in the central zone of the arena, evidenced by an increased number of entries and the distance traveled there. In the EPM, SUC animals spent more time in the open arms and less time in the central square. Metformin treatment prevented the decreased anxiety observed in SUC animals in the OF and EPM. In the NOL test, SUC animals showed less interest in novelty and metformin treatment did not improve this alteration. The preference for open spaces in the OF and EPM were associated with increased serum triglycerides (TG) and malondialdehyde levels in the medial prefrontal cortex (mPFC) and the hippocampus (HIP), while poor memory performance was associated with high basal blood glucose levels. In conclusion, the decreased anxiety-like behavior produced by chronic sucrose consumption was prevented by metformin treatment, through a mechanism that probably involves normalization of TG levels and decreased oxidative stress in mPFC and HIP.

Ultra-High-Resolution Liquid Chromatography Coupled with Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry Analysis of Tessaria absinthioides (Hook. & Arn.) DC. (Asteraceae) and Antioxidant and Hypocholesterolemic Properties.

Recently, we reported the chemical profile and the hypocholesterolemic effects of a decoction of Tessaria absinthioides (Hook. & Arn.) DC. (Asteraceae). In this study, we evaluated a methanolic extract (METa) instead. Metabolite profiling was conducted using ultra-high-resolution liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS), identifying thirty compounds, including flavonoids, phenolic acids, fatty acids, and phorbolesters. Antioxidant properties were assessed through 2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox equivalent antioxidant activity (TEAC), ferric-reducing antioxidant power (FRAP), and inhibition of lipid peroxidation in erythrocytes (ILP) assays, exhibiting robust antioxidant activity. The in vivo impact of METa on serum lipid parameters and liver X receptors (LXRs) was evaluated in a hypercholesterolemic animal model. After 14 days on a high-fat diet, male rats received either a vehicle (V) or METa100, METa200 or METa500 (100; 200 and 500 mg METa/kg animal, respectively) for an additional two weeks. METa500 reduced total cholesterol levels (17.62%; p < 0.05) and all doses increased high-density lipoprotein cholesterol levels (METa100: 86.27%; METa200: 48.37%, and METa500: 29.42%; p < 0.0001). However, METa did not alter LXRs expression. The observed antioxidant and hypocholesterolemic properties of METa may be linked to the presence of six di-caffeoylquinic acids. These findings underscore T. absinthioides as a potential candidate for the treatment of metabolic disease.

Long-Term Memory Function Impairments following Sucrose Exposure in Juvenile versus Adult Rats.

We previously described that excessive consumption of sucrose during youth produces fear memory and anxiety-like behavior in adulthood. Here, we evaluated whether high cognitive function is also affected by studying early sucrose consumption in object recognition memory (NOR). Male Sprague Dawley rats were tested for short-term, long-term, and consolidated NOR after 25 days of unlimited sucrose access in juvenile (PD 25-50) or adult age (PD 75-100). All rats spent equal time exploring the two objects during the sample phase T1. When animals were exposed for 2, 24 h or 7 days later to a copy of the objects presented in T1 and a novel object, the sucrose-exposed juvenile group failed to distinguish between the familiar and the novel objects in contrast with the rest of the groups. Sucrose-exposed animals developed hypertriglyceridemia and glucose intolerance, but juvenile animals showed increased fasting glycemia and sustained the glucose intolerance longer. Moreover, sucrose decreased hippocampal proBDNF expression in juveniles while it was increased in adults, and sucrose also increased RAGE expression in adults. The NOR exploration ratio correlated negatively with basal glycemia and positively with proBDNF. Taken together, these data suggest that sucrose-induced alterations in glucose metabolism may contribute to a long-term decline in proBDNF and impaired recognition memory.

Progesterone and Allopregnanolone Neuroprotective Effects in the Wobbler Mouse Model of Amyotrophic Lateral Sclerosis.

Progesterone regulates a number of processes in neurons and glial cells not directly involved in reproduction or sex behavior. Several neuroprotective effects are better observed under pathological conditions, as shown in the Wobbler mouse model of amyotrophic laterals sclerosis (ALS). Wobbler mice are characterized by forelimb atrophy due to motoneuron degeneration in the spinal cord, and include microgliosis and astrogliosis. Here we summarized current evidence on progesterone reversal of Wobbler neuropathology. We demonstrated that progesterone decreased motoneuron vacuolization with preservation of mitochondrial respiratory complex I activity, decreased mitochondrial expression and activity of nitric oxide synthase, increased Mn-dependent superoxide dismutase, stimulated brain-derived neurotrophic factor, increased the cholinergic phenotype of motoneurons, and enhanced survival with a concomitant decrease of death-related pathways. Progesterone also showed differential effects on glial cells, including increased oligodendrocyte density and downregulation of astrogliosis and microgliosis. These changes associate with reduced anti-inflammatory markers. The enhanced neurochemical parameters were accompanied by longer survival and increased muscle strength in tests of motor behavior. Because progesterone is locally metabolized to allopregnanolone (ALLO) in nervous tissues, we also studied neuroprotection by this derivative. Treatment of Wobbler mice with ALLO decreased oxidative stress and glial pathology, increased motoneuron viability and clinical outcome in a progesterone-like manner, suggesting that ALLO could mediate some progesterone effects in the spinal cord. In conclusion, the beneficial effects observed in different parameters support the versatile properties of progesterone and ALLO in a mouse model of motoneuron degeneration. The studies foresee future therapeutic opportunities with neuroactive steroids for deadly diseases like ALS.

Long-term effects of the glucocorticoid receptor modulator CORT113176 in murine motoneuron degeneration.

The Wobbler mouse spinal cord shows vacuolated motoneurons, glial reaction, inflammation and abnormal glutamatergic parameters. Wobblers also show deficits of motor performance. These conditions resemble amyotrophic lateral sclerosis (ALS). Wobbler mice also show high levels of corticosterone in blood, adrenals and brain plus adrenal hypertrophy, suggesting that chronically elevated glucocorticoids prime spinal cord neuroinflammation. Therefore, we analyzed if treatment of Wobbler mice with the glucocorticoid receptor (GR) antagonist CORT113176 mitigated the mentioned abnormalities. 30 mg/kg CORT113176 given daily for 3 weeks reduced motoneuron vacuolation, decreased astro and microgliosis, lowered the inflammatory mediators high mobility group box 1 protein (HMGB1), toll-like receptor 4, myeloid differentiation primary response 88 (MyD88), p50 subunit of nuclear factor kappa B (NFκB), tumor necrosis factor (TNF) receptor, and interleukin 18 (IL18) compared to untreated Wobblers. CORT113176 increased the survival signal pAKT (serine-threonine kinase) and decreased the death signal phosphorylated Junk-N-terminal kinase (pJNK), symptomatic of antiapoptosis. There was a moderate positive effect on glutamine synthase and astrocyte glutamate transporters, suggesting decreased glutamate excitotoxicity. In this pre-clinical study, Wobblers receiving CORT113176 showed enhanced resistance to fatigue in the rota rod test and lower forelimb atrophy at weeks 2-3. Therefore, long-term treatment with CORT113176 attenuated degeneration and inflammation, increased motor performance and decreased paw deformity. Antagonism of the GR may be of potential therapeutic value for neurodegenerative diseases.

High-Fat Diets and LXRs Expression in Rat Liver and Hypothalamus.

Disturbances on lipid metabolism are associated with health disorders. High-fat diets (HFDs) consumption promotes cardiovascular and neurodegenerative diseases where cholesterol plays an important role. Among regulators of this steroid homeostasis, the liver X receptors (LXRs) induce genes that protect cells from cholesterol overload. We previously described how both hypothalamic LXRα and LXRß are sensitive to a high-fructose diet, suggesting that these receptors trigger responses related to control of energy and food intake. The present work's main objective was to study the effect of different HFDs on LXRs expression (in hypothalamus and liver), and lipid profile. Male rats received control diet (CD), HFD1 (CD + bovine fat (BF)), HFD2 (CD + BF + cholic acid (CA)), HFD3 (CD + BF + cholesterol), or HFD4 (CD + BF + CA + cholesterol) for different time periods. Hypothalamic LXRß, both hepatic LXRs subtypes, and total cholesterol (TC) raised after 2 weeks of HFDs. Four and 8 weeks of HFD3 and HFD4 increased the LXRs subtypes in both tissues and TC levels. Only HFD4 reduced triglycerides (TG) levels after 2 and 8 weeks. The TC and TG values correlated significantly with LXRs expression only in rats fed with HFD4. These data add relevant information about how diet composition can produce different scales of hypercholesterolemia states accompanied with central and peripheral changes in the LXRs expression.

Sucrose exposure in juvenile rats produces long-term changes in fear memory and anxiety-like behavior.

Sugar consumption has increased dramatically in our society, a phenomenon that is primarily associated with obesity and diabetes appearance. However, whether this overconsumption of sugar has an impact on the developing CNS remains unknown. This study investigated the long-term effects of unlimited access to sucrose using the two-bottle choice paradigm and the juvenile and adult effects were compared. Male Sprague Dawley rats had free access to water containing 10% sucrose and water during youth (PD 25-50) or adulthood (PD 75-100). Rats in the sucrose group, privileged to take sugary solution over the water. No weight differences were observed between the sucrose groups and their age-matched water controls. After treatment all animals drank only water for another 25 days. Frustration, measured as the amount of water drank after the sucrose period, was higher in young-exposed animals compared to adults. In addition, rats that consumed sucrose during youth travelled less the central zones of an open field. Sucrose consumption during youth also affected fear behavior as animals exhibited impaired extinction of fear memory compared to control, indicating that prefrontal and hippocampal function is impaired. In contrast, rats exposed to sucrose during adulthood did not behave significantly different from control on either task. The calretinin and parvalbumin GABAergic interneurons go through extensive remodeling during youth in the medial prefrontal cortex and the ventral hippocampus. Here, we found that rats exposed to sucrose during youth presented an increased expression of calretinin-immunoreactivity in the medial prefrontal cortex, but not in the ventral hippocampus, indicating that early sucrose consumption produces enduring effects on the GABA system. Altogether these results indicate that sugar overconsumption at early stages of life induces long-term effects on behaviors related to fear and anxiety in adulthood.

Severe Uncontrolled Maternal Hyperglycemia Induces Microsomia and Neurodevelopment Delay Accompanied by Apoptosis, Cellular Survival, and Neuroinflammatory Deregulation in Rat Offspring Hippocampus.

Maternal diabetes constitutes an unfavorable intrauterine environment for offspring development. Although it is known that diabetes can cause brain alterations and increased risk for neurologic disorders, the relationship between neuroimmune activation, brain changes, and neurodevelopment deficits in the offspring remains unclear. In order to elucidate the short- and long-term biological basis of the developmental outcomes caused by the severe uncontrolled maternal hyperglycemia, we studied apoptosis, neurogenesis, and neuroinflammation pathways in the hippocampus of neonates and young rats born to diabetic dams. Diabetes was induced on gestational day 5 by an injection of streptozotocin. Evaluations of milestones, body growth, and inhibitory avoidance were performed to monitor the offspring development and behavior. Hippocampal modifications were studied through cellular survival by BrdU in the dentate gyrus, expression of apoptosis-regulatory proteins (procaspase 3, caspase 3, and Bcl-2), BDNF, and neuroinflammatory modulation by interleukins, MHC-I, MHC-II, Iba-1, and GFAP proteins. Severe maternal diabetes caused microsomia and neurodevelopmental delay in pups and decrease of Bcl-2, procaspase 3, and caspase 3 in the hippocampus. Moreover, in a later stage of development, it was found an increase of TNF-α and a decrease of procaspase 3, caspase 3, MHC-I, IL-1ß, and BDNF in the hippocampus, as well as impairment in cellular survival in the dentate gyrus. This study showed significant short- and long-term commitments on the development, apoptosis, cell survival, and neuroinflammation in the offspring hippocampus induced by severe uncontrolled maternal hyperglycemia. The data reinforce the need for treatment of maternal hyperglycemic states during pregnancy and breast-feeding.

LXR activation increases the expression of GnRH AND αMSH in the rat hypothalamus in vivo.

Liver X receptors (LXR) are important transcription factors involved in the regulation of carbohydrate and lipid metabolism. Recently, we described LXR receptors expression in the hypothalamus but their function in this brain area remains unknown. Here, we evaluated the function of LXR on the expression of factors produced in the hypothalamus in vitro and in vivo by Western blotting and immunocytochemistry. More precisely we studied the expression of GnRH and GHRH, αMSH and NPY in male Sprague-Dawley rats. The effects of two synthetic LXR agonists, T0901317 and GW3965, were first tested in vitro. Hypothalamic explants were treated with either T0901317 or GW3965 (10µM) for 2, 4, 6 and 8h. As a positive control the cholesterol ABCA1 and glucose GLUT2 transporters were used. No changes were observed in the expression of the factors evaluated in vitro. The effects of the LXR agonists were then tested in vivo. Rats were injected ICV into the third ventricle with either T0901317 or GW3965 (2.5µg/5µL ICV) and after 3.5h or 24h the hypothalami were dissected out and rapidly frozen for analysis. αMSH and GnRH expression was significantly increased after 3.5h of T0901317 treatment. Anterior/posterior hypothalamic ratio increases for αMSH expression and decreases for GnRH expression after 24h of LXR activation. Altogether these results show that LXR activation affects the expression of GnRH and αMSH, suggesting that LXR in the hypothalamus is capable of modulating hypothalamic responses related to appetite, sexual behavior and reproductive functions.

Protective effects of the neurosteroid allopregnanolone in a mouse model of spontaneous motoneuron degeneration.

Amyotrophic lateral sclerosis (ALS) is a devastating disorder characterized by progressive death of motoneurons. The Wobbler (WR) mouse is a preclinical model sharing neuropathological similarities with human ALS. We have shown that progesterone (PROG) prevents the progression of motoneuron degeneration. We now studied if allopregnanolone (ALLO), a reduced metabolite of PROG endowed with gabaergic activity, also prevents WR neuropathology. Sixty-day old WRs remained untreated or received two steroid treatment regimens in order to evaluate the response of several parameters during early or prolonged steroid administration. ALLO was administered s.c. daily for 5days (4mg/kg) or every other day for 32days (3, 3mg/kg), while another group of WRs received a 20mg PROG pellet s.c. for 18 or 60days. ALLO administration to WRs increased ALLO serum levels without changing PROG and 5 alpha dihydroprogesterone (5α-DHP), whereas PROG treatment increased PROG, 5α-DHP and ALLO. Untreated WRs showed higher basal levels of serum 5α-DHP than controls. In the cervical spinal cord we studied markers of oxidative stress or associated to trophic responses. These included nitric oxide synthase (NOS) activity, motoneuron vacuolation, MnSOD immunoreactivity (IR), brain derived neurotrophic factor (BDNF) and TrkB mRNAs, p75 neurotrophin receptor (p75NTR) and, cell survival or death signals such as pAKT and the stress activated kinase JNK. Untreated WRs showed a reduction of MnSOD-IR and BDNF/TrkB mRNAs, associated to high p75NTR in motoneurons, neuronal and glial NOS hyperactivity and neuronal vacuolation. Also, low pAKT, mainly in young WRs, and a high pJNK in the old stage characterized WRs spinal cord. Except for MnSOD and BDNF, these alterations were prevented by an acute ALLO treatment, while short-term PROG elevated MnSOD. Moreover, after chronic administration both steroids enhanced MnSOD-IR and BDNF mRNA, while attenuated pJNK and NOS in glial cells. Long-term PROG also increased pAKT and reduced neuronal NOS, parameters not modulated by chronic ALLO. Clinically, both steroids improved muscle performance. Thus, ALLO was able to reduce neuropathology in this model. Since high oxidative stress activates p75NTR and pJNK in neurodegeneration, steroid reduction of these molecules may provide adequate neuroprotection. These data yield the first evidence that ALLO, a gabaergic neuroactive steroid, brings neuroprotection in a model of motoneuron degeneration.

Regulation of the expression of LXR in rat hypothalamic and hippocampal explants.

Liver X receptors (LXR) are important transcription factors involved in the regulation of carbohydrate and lipid metabolism and are expressed in different brain areas. Recently we described that LXR expression in the hypothalamus is sensitive to serum levels of lipids and carbohydrates. Here, we further characterized the effects of glucose, insulin, cholesterol and cholic acid on the expression of LXRα and LXRß in hypothalamus and hippocampus explants as in vitro models. The LXR activation products, GLUT2 and ABCA1, were also analyzed by Western blot. Glucose had different effects in the hypothalamus compared to the hippocampus. In the hypothalamus, increases in glucose concentrations decreased LXRß expression while in the hippocampus increased both receptor subtypes levels. In contrast, insulin treatment decreased LXRß in the hypothalamus while having no effects on the hippocampus. Cholic acid and cholesterol increased only LXRα expression in the hypothalamus whereas no effects on the hippocampus were detected. The newly expressed LXR receptors may be functional active since the level of the LXR activation product ABCA1 was also increased. Changes in GLUT2 expression was observed only when LXRß levels were increased. Altogether these data show that LXR are sensitive to glucose, insulin and lipids in vitro, as well as in vivo as we previously showed, suggesting an involvement of LXR in central metabolic pathways and control of energy homeostasis.

Sex differences in LXR expression in normal offspring and in rats born to diabetic dams.

Gestational diabetes (GD) alters normal fetal development and is related to a diabetogenic effect in the progeny. Liver X receptors (LXRs) are considered to be potential drug targets for the regulation, treatment, or prevention of diabetes. The aim of this study was to evaluate early and late changes of LXR in the hippocampus and hypothalamus of the male and female offspring of control (CO) and diabetic (DO) mothers. We used an experimental model of streptozotocin-induced GD to assess the protein expression of LXRα (NR1H3) and LXRß (NR1H2) by western blotting. The tissues were obtained from CO and DO animals at postnatal day 1 (1D), day 10 (10D), and day 35 (35D) and 9 months (9M). In CO, the LXR expression showed significant differences among the groups, which were tissue- and receptor-specific (P<0.05). Sex differences in CO were found only in the hypothalamus for LXRß expression at 35D and 9M (P<0.05). When CO and DO were compared, differences between them were observed in the majority of the studied groups at 1D (male hippocampus, LXRα 31% and LXRß 161%; female hippocampus, LXRß 165%; male hypothalamus, LXRß 182%; and female hypothalamus, LXRα 85%; P<0.05). However, these differences disappeared later with the exception of LXRß expression in the male hypothalamus (P<0.05). The area under the curve during the glucose tolerance test correlated negatively with LXRß in CO but not in DO animals. Moreover, in a male DO subpopulation this correlation was positive as it occurs in intolerant animals. These results indicate that GD affects hypothalamic LXR expression differently in male and female offspring.

Neuroprotective action of synthetic steroids with oxygen bridge. Activity on GABAA receptor.

Allopregnanolone (A) and pregnanolone (P) are able to modify neural activities acting through the GABAA receptor complex. This capacity makes them useful as anticonvulsant, anxiolytic, or anti-stress compounds. In this study, the performance of seven synthetic steroids (SS) analogous of A or P containing an intramolecular oxygen bridge was evaluated using different assays. Competition assays showed that compounds 1, 5, 6 and 7 affected the binding of specific ligands for the GABAA receptor in a way similar to that of A and P, whereas compounds 3 and 4 stimulated [(3)H]-flunitrazepam and reduced [(35)S]-TBPS binding. The enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD) produces the precursor for A and P, and its activity is regulated by steroids. The action of several SS on 3ß-HSD activity was tested in different tissues. All SS analyzed inhibit its activity, but compound 5 was the least effective. Finally, the neuroprotective role of two SS was evaluated in cerebral cortex and hippocampus cultures subjected to hypoxia. Glial fibrillary acidic protein (GFAP) increase was prevented by A, P, and compounds 3 and 5. Only A, P and compound 5 prevented neurofilament (NF160/200) decrease in hippocampus cultures, whereas A and compound 5 partially prevented NF200 and NF160 decreases respectively in cerebral cortex cultures. A prevented microtubule associated protein (MAP 2b) decrease in cerebral cortex cultures, while in hippocampus cultures only compounds 3 and 5 had effect. All steroids prevented MAP 2c decrease in both brain regions.

Alterations of LXRα and LXRß expression in the hypothalamus of glucose-intolerant rats.

Liver X receptor (LXR) α and ß are nuclear receptors that are crucial for the regulation of carbohydrate and lipid metabolism. Activation of LXRs in the brain facilitates cholesterol clearance and improves cognitive deficits, thus they are considered as promising drug targets to treat diseases such as atherosclerosis and Alzheimer's disease. Nevertheless, little is known about the function and localization of LXRs in the brain. Here, we studied the expression of LXR in the brains of rats that received free access to 10% (w/v) fructose group (FG) in their beverages or water control drinks (control group (CG)). After 6 weeks rats in the FG presented with hypertriglyceridemia, hyperinsulinemia, and became glucose intolerant, suggesting a progression toward type 2 diabetes. We found that hypothalamic LXR expression was altered in fructose-fed rats. Rats in the FG presented with a decrease in LXRß levels while showing an increase in LXRα expression in the hypothalamus but not in the hippocampus, cerebellum, or neocortex. Moreover, both LXRα and ß expression correlated negatively with insulin and triglyceride levels. Interestingly, LXRß showed a negative correlation with the area under the curve during the glucose tolerance test in the CG and a positive correlation in the FG. Immunocytochemistry revealed that the paraventricular and ventromedial nuclei express mainly LXRα whereas the arcuate nucleus expresses LXRß. Both LXR immunosignals were found in the median preoptic area. This is the first study showing a relationship between glucose and lipid homeostasis and the expression of LXRs in the hypothalamus, suggesting that LXRs may trigger neurochemical and neurophysiological responses for the control of food intake and energy expenditure through these receptors.

Down regulation of the proliferation and apoptotic pathways in the embryonic brain of diabetic rats.

Compelling evidence shows that the offspring subjected to uncontrolled hyperlycemia during gestation display behavioral, neurochemical, and cellular abnormalities during adulthood. However, the molecular mechanisms underlying these defects remain elusive. Previous studies have shown an increased rate of apoptosis and a decreased index of neuronal proliferation associated with diabetic embryopathy. The aim of the present study was to determine whether impairments in apoptotic related proteins also occur in the developing central nervous system from non-malformed embryos exposed to uncontrolled gestational hyperglycemia. Pregnant rats injected with either streptozotocin or vehicle were killed on gestational day 19. Offspring brains were quickly removed to evaluate protein expression by Western blotting. Embryonic brains from diabetic rats exhibited a decrease in the cell survival p-Akt expression (52.83 ± 24.35%) and in the pro-apoptotic protein Bax (56.16 ± 6.47%). Moreover, the anti-apoptotic protein Bcl-2 showed a non-significant increase while there were no changes in Procaspase 3 or cleaved Caspase 3 proteins. The cytoskeleton proteins NF-200 and GFAP were also examined. Neither NF-200 nor GFAP showed differences in embryonic brains from diabetic rats compared to controls. Altogether, these results indicate that both proliferation and apoptotic pathways are decreased in the brain from the developing offspring of diabetic rats. Since selective neuronal apoptosis, as well as selective cell proliferation, are specifically involved in brain organogenesis, it is possible that simultaneous impairments during the perinatal period contribute to the long lasting alterations observed in the adult brain.

Allopregnanolone effects on astrogliosis induced by hypoxia in organotypic cultures of striatum, hippocampus, and neocortex.

UNLABELLED: Perinatal asphyxia occurs in approximately 0.3% full-term newborn babies, and this percentage has not decreased despite medical advances. There are now evidences indicating that neurosteroids are important in neurodevelopment showing neuroprotective effects. We studied the potential protective effect of allopregnanolone (Allo) in vitro using organotypic cultures from neocortex, striatum, and hippocampus. Immunocytochemistry and confocal microscopy showed an increase of the glial fibrillary acidic protein (GFAP) signal in the studied brain areas after hypoxia. Western blot studies supported these results (hippocampus, 193%; neocortex, 306%; and striatum, 231%). Twenty-four-hour pretreatment with Allo showed different effects at the brain areas studied. In the hippocampus and the neocortex, 24-h pretreatment with Allo 5x10(-6) M showed to be neuroprotective as there was a significant decrease of the GFAP signal compared to control cultures exposed to hypoxia. Pretreatment with 5x10(-8) M Allo attenuated the astrogliosis response in the hippocampus and the neocortex in a nonsignificant way. Allo pretreatment at all doses did not show to affect the astrogliosis triggered by hypoxia in the striatum. Cell survival was analyzed by measuring LDH. After 1 h of hypoxia, all cultures showed a nonsignificant increase of LDH, which was greater after 24 h of hypoxia (hippocampus, 180%; striatum-cortex co-cultures, 140%). LDH levels have no changes by Allo pretreatment before hypoxia. CONCLUSION: 24 h pretreatment with 5x10(-6) M of Allo does not change neuronal viability but it prevents astrogliosis induced by hypoxia in the hippocampus and the neocortex.

Interaction of dopamine D1 with NMDA NR1 receptors in rat prefrontal cortex.

Despite the tremendous importance of D1 and NMDA receptors to cognition (working memory, executive functions) and synaptic plasticity in the prefrontal cortex (PFC), little is known about the molecular mechanisms underlying D1-NMDA receptors interactions in this brain area. Here, we show that D1 receptors and the NMDA receptor co-localize in single pyramidal neurons and interneurons in adult rat PFC. NR1 and NR2A expression are found in different neuronal types. Conversely, D1 receptors are predominantly localized in pyramidal-like cells and parvalbumin positive cells. NR1 co-immunoprecipitates with D1 receptor in adult medial PFC. In prefrontal primary cultures, NMDA does not affect the D1 receptor dependent-cAMP production. In contrast, activation of D1 receptor potentiates the NMDA mediated increase in cytosolic Ca2+, an effect that was blocked by a PKA inhibitor. We conclude that D1 receptor potentiates the NMDA-Ca2+ signal by a PKA-dependent mechanism.

A tridimensional view of the organization of actin filaments in the central nervous system by use of fluorescent photooxidation.

Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions.

Recruitment of renal dopamine 1 receptors requires an intact microtubulin network.

Renal dopamine1 receptor (D1R) can be recruited from intracellular compartments to the plasma membrane by D1R agonists and endogenous dopamine. This study examines the role of the cytoskeleton for renal D1R recruitment. The studies were performed in LLCPK-1 cells that have the capacity to form dopamine from L-dopa. In approximately 50% of the cells treated with L-dopa the D1R was found to be translocated from intracellular compartments towards the plasma membrane. Disruption of the microtubulin network by nocodazole significantly prevented translocation. In contrast, depolymerization of actin had no effect. In control cells D1R colocalized with NBD-C(6)-ceramide, a trans-Golgi fluorescent marker. This colocalization was disrupted in L-dopa-treated cells. Tetanus toxin, an inhibitor of exocytosis, prevented L-dopa-induced receptor recruitment. L-Dopa treatment resulted in activation of protein kinase C (PKC). To test the functional effect of D1R recruitment, the capacity of D1R agonists to activate PKC was studied. Activation of D1R significantly translocated PKC-alpha from intracellular compartments to the plasma membrane. Disruption of microtubules abolished D1R-mediated - but not phorbol-ester-mediated - translocation of PKC. We conclude that renal D1R recruitment requires an intact microtubulin network and occurs via Golgi-derived vesicles. These newly recruited receptors couple to the PKC signaling pathway.