Claudia Grillo

Memory impairment in obese Zucker rats: an investigation of cognitive function in an animal model of insulin resistance and obesity.

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.

Peripheral glucose administration stimulates the translocation of GLUT8 glucose transporter to the endoplasmic reticulum in the rat 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. J. Comp. Neurol. 452:103–114, 2002.

Region specific increases in oxidative stress and superoxide dismutase in the hippocampus of diabetic rats subjected to stress

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, Ammons 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.

Biphasic effects of stress upon GLUT8 glucose transporter expression and trafficking in the diabetic rat hippocampus.

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.

Involvement of Mesolimbic Structures in Short-Term Sodium Depletion: In situ Hybridization and Ligand-Binding Analyses

Acute treatment with the diuretic furosemide (Lasix) produces a reduction in plasma Na+ and volume as well as increased thirst and salt appetite. The resulting hypovolemia stimulates the well-known counter-regulatory physiological response from the renin-angiotensin-aldosterone system. However, the neurochemical players underpinning the behavioral responses of thirst and salt appetite are less clear. Previously, we have reported that salt-replete deoxycorticosterone (DOCA) treatment activates mesolimbic structures associated with reward and goal-seeking behavior. The present study was designed to test whether the same brain regions are affected in a salt-depleted state. In experiment 1, two groups of adult male Sprague-Dawley (SD) rats were injected with Lasix (10 mg/rat, s.c.) and 18 h later were allowed access either to 2% NaCl solution (‘Lasix+salt’) or only to tap water (‘Lasixnosalt’) for 2 h. For comparison purposes, a third group received an isotonic saline injection instead of Lasix and was allowed access to the 2% salt solution (Vehicle). All groups were permitted 24 h access to tap water. We found no differences in dynorphin-mRNA levels in any striatal and accumbal regions among any of the treatment groups. However, as found previously in DOCA-treated rats, there were increased enkephalin (ENK)-mRNA and decreased dopamine transporter (DAT) binding levels throughout the striatum in Lasix+salt and decreased ENK-mRNA in Lasixnosalt rats versus Vehicle. In experiment 2, the involvement of the ENKergic and/or dopaminergic system was tested in rats divided into the same three groups described in experiment 1. However, before access to salt or water, the Lasix+salt and the vehicle groups were administered either a δ-opioid, naltrindole or a dopamine D2 antagonist, raclopride. Only the naltrindole-treated rats showed a blunted intake of salt solution. Thus, these findings along with our neurochemical results suggest that mesolimbic enkephalin might impact salt intake through dopaminergic systems.

Further Studies in Deoxycorticosterone Acetate Treated Rats: Brain Content of Mineralocorticoid and Glucocorticoid Receptors and Effect of Steroid Antagonists on Salt Intake

We have studied the role of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) on salt appetite developed by deoxycorticosterone acetate (DOCA) treated rats. To this end, we measured the effects of DOCA given on alternate days on (1) salt intake; (2) MR and GR in hippocampus (HIPPO), amygdala (AMYG), and hypothalamus (HT); (3) the activity of ornithine decarboxylase (ODC), a GR-mediated response, and (4) the salt intake after treatment with the antiglucocorticoid RU 486 or the antimineralocorticoid ZK 91587. First, we demonstrated that 10 but not 1 mg DOCA induced natriogenesis. Forty-eight hours after adrenalectomy and 24 h after the last DOCA injection, 10 but not 1 mg hormone reduced binding to GR in HIPPO, AMYG, and HT. Both doses of DOCA also reduced the binding to MR in HIPPO, without changes in AMYG; in HT the 1-mg dose was without effect, but the natriogenic dose (10 mg) highly increased binding of [3H]-corticosterone to MR. Scatchard analysis demonstrated increased Bmax and Kd values in the HT of DOCA-treated rats. Occupation of GR by DOCA did not stimulate the ODC activity, in contrast to the four-fold increment effected by the glucocorticoid dexamethasone. Also, administration of RU 486 did not inhibit the sale intake promoted by DOCA, in contrast to ZK 91587 which partly delayed the natriogenic effect of DOCA. It is suggested that brain MR are involved in the natriogenic effect of DOCA, whereas the role of GR is inconclusive.(ABSTRACT TRUNCATED AT 400 WORDS)

Sex Difference in Glucocorticoid Regulation of Vasopressin mRNA in the Paraventricular Hypothalamic Nucleus

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

Increased Expression of Magnocellular Vasopressin mRNA in Rats with Deoxycorticosterone-Acetate Induced Salt Appetite

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.

Glucocorticoid Regulation of Glycerol Phosphate Dehydrogenase and Ornithine Decarboxylase Activities in the Spinal Cord of the Rat

Abstract: We examined the effects of glucocorticoids on induction of glycerol phosphate dehydrogenase (GPDH) and ornithine decarboxylase (ODC) in the spinal cord of rats. After a single subcutaneous dose of 5 mg/kg of dexametha‐sone (DEX) phosphate, GPDH activity was maximally increased at 20 h with the effect still persisting for 46 h, in contrast to ODC activity, which was already stimulated at 4 h. The enzyme induction was accompanied by a reduction in number of cytosolic glucocorticoid receptors already at 1 h after DEX treatment, with replenishment at 22 h. A dose‐response curve for DEX demonstrated that the minimal effective dose (0.2 mg/kg) for enzyme induction also reduced the number of cytosolic receptors because of occupation/depletion. The effects were specific for natural and synthetic glucocorticoids, as GPDH and ODC activities were not stimulated by aldosterone, testosterone, estradiol, or progesterone. ODC was induced in the cervical region of the spinal cord as well as in the horse tail plus filum terminale, whereas GPDH responded in the former but not the latter region. Previous work has demonstrated that glucocorticoid receptors are slightly more concentrated in the cervical spinal cord. It is suggested that glucocorticoid induction of these two predominantly glial enzymes occurs by a steroid receptor‐mediated event, as postulated in other regions of the nervous system. In view of the short latency required for induction of ODC, we also examined the effect of inhibitors of transcription and translation. Whereas cycloheximide reduced the stimulatory effect of DEX, a paradoxical stimulation was obtained when DEX and dactinomycin (actinomy‐cin D) were given concomitantly. It is suggested that the inductive responses of GPDH and ODC to glucocorticoids may be different. Considering that GPDH induction has been shown by other laboratories to represent a genomic effect of adrenal steroids, different levels of control may account for the stimulation of ODC in the spinal cord.

Chronic stress alters amphetamine effects on behavior and synaptophysin levels in female rats

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.