Mary R. DeJoseph

Na+-dependent Glutamate Transporters (EAAT1, EAAT2, and EAAT3) of the Blood-Brain Barrier A MECHANISM FOR GLUTAMATE REMOVAL

Na+-dependent transporters for glutamate exist on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3). These transporters presumably assist in keeping the glutamate concentration low in the extracellular fluid of brain. Recently, Na+-dependent glutamate transport was described on the abluminal membrane of the blood-brain barrier. To determine whether the above-mentioned transporters participate in glutamate transport of the blood-brain barrier, total RNA was extracted from bovine cerebral capillaries. cDNA for EAAT1, EAAT2, and EAAT3 was observed, indicating that mRNA was present. Western blot analysis demonstrated all three transporters were expressed on abluminal membranes, but none was detectable on luminal membranes of the blood-brain barrier. Measurement of transport kinetics demonstrated voltage dependence, K+-dependence, and an apparentK m of 14 μm (aggregate of the three transporters) at a transmembrane potential of −61 mV. Inhibition of glutamate transport was observed using inhibitors specific for EAAT2 (kainic acid and dihydrokainic acid) and EAAT3 (cysteine). The relative activity of the three transporters was found to be approximately 1:3:6 for EAAT1, EAAT2, and EAAT3, respectively. These transporters may assist in maintaining low glutamate concentrations in the extracellular fluid.

Positive, But Not Negative Feedback Actions of Estradiol in Adult Female Mice Require Estrogen Receptor α in Kisspeptin Neurons

Hypothalamic kisspeptin (Kiss1) neurons express estrogen receptor α (ERα) and exert control over GnRH/LH secretion in female rodents. It has been proposed that estradiol (E2) activation of ERα in kisspeptin neurons in the arcuate nucleus (ARC) suppresses GnRH/LH secretion (negative feedback), whereas E2 activation of ERα in kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) mediates the release of preovulatory GnRH/LH surges (positive feedback). To test these hypotheses, we generated mice bearing kisspeptin cell-specific deletion of ERα (KERαKO) and treated them with E2 regimens that evoke either negative or positive feedback actions on GnRH/LH secretion. Using negative feedback regimens, as expected, E2 effectively suppressed LH levels in ovariectomized (OVX) wild-type (WT) mice to the levels seen in ovary-intact mice. Surprisingly, however, despite the fact that E2 regulation of Kiss1 mRNA expression was abrogated in both the ARC and AVPV of KERαKO mice, E2 also effectively decreased LH levels in OVX KERαKO mice to the levels seen in ovary-intact mice. Conversely, using a positive feedback regimen, E2 stimulated LH surges in WT mice, but had no effect in KERαKO mice. These experiments clearly demonstrate that ERα in kisspeptin neurons is required for the positive, but not negative feedback actions of E2 on GnRH/LH secretion in adult female mice. It remains to be determined whether the failure of KERαKO mice to exhibit GnRH/LH surges reflects the role of ERα in the development of kisspeptin neurons, in the active signaling processes leading to the release of GnRH/LH surges, or both.

Different Roles of BDNF in Nucleus Accumbens Core versus Shell during the Incubation of Cue-Induced Cocaine Craving and Its Long-Term Maintenance

Brain-derived neurotrophic factor (BDNF) contributes to diverse types of plasticity, including cocaine addiction. We investigated the role of BDNF in the rat nucleus accumbens (NAc) in the incubation of cocaine craving over 3 months of withdrawal from extended access cocaine self-administration. First, we confirmed by immunoblotting that BDNF levels are elevated after this cocaine regimen on withdrawal day 45 (WD45) and showed that BDNF mRNA levels are not altered. Next, we explored the time course of elevated BDNF expression using immunohistochemistry. Elevation of BDNF in the NAc core was detected on WD45 and further increased on WD90, whereas elevation in shell was not detected until WD90. Surface expression of activated tropomyosin receptor kinase B (TrkB) was also enhanced on WD90. Next, we used viral vectors to attenuate BDNF-TrkB signaling. Virus injection into the NAc core enhanced cue-induced cocaine seeking on WD1 compared with controls, whereas no effect was observed on WD30 or WD90. Attenuating BDNF-TrkB signaling in shell did not affect cocaine seeking on WD1 or WD45 but significantly decreased cocaine seeking on WD90. These results suggest that basal levels of BDNF transmission in the NAc core exert a suppressive effect on cocaine seeking in early withdrawal (WD1), whereas the late elevation of BDNF protein in NAc shell contributes to incubation in late withdrawal (WD90). Finally, BDNF protein levels in the NAc were significantly increased after ampakine treatment, supporting the novel hypothesis that the gradual increase of BDNF levels in NAc accompanying incubation could be caused by increased AMPAR transmission during withdrawal.

Neomycin Reduces the Intestinal Production of Ammonia from Glutamine

The mechanism by which neomycin treatment reduces circulating ammonia concentrations was studied in normal and portacaval shunted rats. Rats were given neomycin for 3 days and then fasted for 24 hours to eliminate feces. Neomycin decreased arteriovenous differences of ammonia across the intestine even when the intestines were empty. Neomycin treatment lowered the activity of glutaminase in the intestinal mucosa and the rate of ammonia production from glutamine by isolated intestinal segments. The intestines from portacaval shunted rats had higher glutaminase activity (by 57%), and produced ammonia from glutamine at a greater rate (by 31%), than intestines from controls. Neomycin treatment lowered glutaminase activity and ammonia production in shunted rats, but glutaminase activity still remained higher than in controls (by 23%). The data indicate that the mechanism by which neomycin lower plasma ammonia is owing, at least in part, to a direct effect on the intestines. Specifically, neomycin causes a reduction in mucosal glutaminase activity and thereby decreases the ability of the mucosa to consume glutamine and produce ammonia.

Glucose transporters and glucose utilization in rat brain after acute ethanol administration.

In the normal adult brain, glucose provides 90% of the energy requirements as well as substrate for nucleic acid and lipid synthesis. In the present study, effects of ethanol on glucose transporters (GLUT) and glucose utilization were examined in rat brain. Male Sprague-Dawley rats weighing 250–300 gms were given either ethanol 3 gm/kg BW or saline IP 4 hrs prior to the animal sacrifice and removal of the cerebral cortical tissue. The cortical plasma membranes analyzed by cytochalasin B binding assay showed a decrease in GLUT number but not in GLUT affinity in the ethanol treated rats as compared to the control rats. The estimated Ro values were 70±8.9 Vs 91±8.9 pmoles/mg protein (p<0.05 N=4) and the estimated Kd values were 0.37±0.03 and 0.28±0.05 μM (p: NS) in ethanol and control experiments respectively. Immunoblots of purified cerebral plasma membranes and low density microsomal fraction showed 17% and 71% decrease for GLUT1 and 54% and 21% (p<0.05 or less; n=6) for GLUT3 respectively in ethanol treated rats than in control animals. Immunofluoresence studies also showed reduction of GLUT1 immunoreactively in choroid plexus and cortical microvessels of ethanol treated rats as compared to control rats. The effect of ethanol on regional cerebral metabolic rates for glucose (CMRGk) was studied using [6-14C] glucose and showed statistically insignificant decrease in brain glucose, utilization. These data suggest that ethanol invivo decrease GLUT number and protein content in rat cerebral cortex

Regional brain monoamines and their metabolites after portacaval shunting.

Disturbances in brain monoamine neurotransmitter metabolism have been implicated in the development of hepatic encephalopathy produced by portacaval shunting or liver disease. We have measured the content of serotonin, norepinephrine and dopamine, as well as their metabolites 5-hydroxyindoleacetic acid, dihydroxyphenylacetic acid and homovanillic acid in nine selected brain areas of rats with portacaval shunts and sham-operated control rats. All substances were measured in single samples using high performance liquid chromatography with electrochemical detection, after a simple extraction procedure. In shunted rats serotonin content was 26% higher in the raphe nuclei area, and 5-hydroxyindoleacetic acid throughout the brain (by 51 to 137%), suggesting increased serotonin turnover. Norepinephrine content was higher by 26% in the frontal cortex. Dopamine content was unaffected; however its metabolites were higher in a few areas including the caudate and ventral tegmentum. Brain content of the monoamine precursor amino acids tryptophan, tyrosine and phenylalanine was higher throughout the brain in the shunted rats. The results suggest that serotonin metabolism is altered throughout the brain after portacaval shunting, which could be related to some of the characteristic behavioral abnormalities found in this condition. Catecholamine metabolism appears to be more selectively and less extensively affected.

Glutamate permeability at the blood-brain barrier in insulinopenic and insulin-resistant rats

The influence of diabetes on brain glutamate (GLU) uptake was studied in insulinopenic (streptozotocin [STZ]) and insulin-resistant (diet-induced obesity [DIO]) rat models of diabetes. In the STZ study, adult male Sprague-Dawley rats were treated with STZ (65 mg/kg intravenously) or vehicle and studied 3 weeks later. The STZ rats had elevated plasma levels of glucose, ketone bodies, and branched-chain amino acids; brain uptake of GLU was very low in both STZ and control rats, examined under conditions of normal and greatly elevated (by intravenous infusion) plasma GLU concentrations. In the DIO study, rats ingested a palatable, high-energy diet for 2 weeks and were then divided into weight tertiles: rats in the heaviest tertile were designated DIO; rats in the lightest tertile, diet-resistant (DR); and rats in the intermediate tertile, controls. The DIO and DR rats continued to consume the high-energy diet for 4 more weeks, whereas the control rats were switched to standard rat chow. All rats were studied at 6 weeks (subgroups were examined under conditions of normal or elevated plasma GLU concentrations). The DIO rats ate more food and were heavier than the DR or control rats and had higher plasma leptin levels and insulin-glucose ratios. In all diet groups, the blood-brain barrier showed very low GLU penetration and was unaffected by plasma GLU concentration. Brain GLU uptake also did not differ among the diet groups. Together, the results indicate that the blood-brain barrier remains intact to the penetration of GLU in 2 models of diabetes under the conditions examined.

Diurnal rhythm returns to normal after elimination of portacaval shunting

Previous studies showed that portacaval shunting causes metabolic and behavioral changes in rats. Most metabolic changes reversed within 1-2 wk after restoration of normal circulation. However, the rate of cerebral glucose metabolism (CMRGlc) remained depressed in some areas. The question arose whether complete recovery was possible. Therefore, a long-term behavioral study was undertaken to determine the time course of recovery. Diurnal activity was monitored for 48 h each week over a period of 14 wk: 2 wk before shunting, 6 wk after shunting, and 6 wk after restoration of normal hepatic circulation. Nighttime activity was depressed within 1 wk of shunting and did not change. Normal circulation to the liver was reestablished after 6 wk. The diurnal cycle was normal 3 wk later. Thus, although recovery of the diurnal rhythm is possible, the relatively long period necessary suggests the correction of a significant structural or chemical abnormality. A study of CMRGlc was made using the behavioral study as an index of the time necessary for recovery. CMRGlc returned to normal throughout the brain 6 wk after cessation of shunting except in the hippocampus and amygdala (7-8% decrease).