Obesity induced by neonatal treatment with monosodium glutamate impairs microvascular reactivity in adult rats: Role of NO and prostanoids

Background and aim: given that obesity is an independent risk factor for the development of cardiovascular diseases we decided to investigate the mechanisms involved in microvascular dysfunction using a monosodium glutamate (MSG)-induced model of obesity, which allows us to work on both normotensive and normoglycemic conditions. Methods and results: Male offspring of Wistar rats received MSG from the second to the sixth day after birth. Sixteen-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia and insulin resistance, with no alteration in glycemia and blood pressure. The effect of norepinephrine (NE), which was increased in MSG rats, was potentiated by L-nitro arginine methyl ester (L-NAME) or tetraethylammonium (TEA) and was reversed by indomethacin and NS-398. Sensitivity to acetylcholine (ACh), which was reduced in MSG rats, was further impaired by L-NAME or TEA, and was corrected by indomethacin, NS-398 and tetrahydrobiopterin (BH4). MSG rats displayed increased endothelium-independent relaxation to sodium nitroprusside. A reduced prostacyclin/tromboxane ratio was found in the mesenteric beds of MSG rats. Mesenteric arterioles of MSG rats also displayed reduced nitric oxide (NO) production along with increased reactive oxygen species (ROS) generation; these were corrected by BH4 and either L-NAME or superoxide dismutase, respectively. The protein expression of eNOS and cyclooxygenase (COX)-2 was increased in mesenteric arterioles from MSG rats. Conclusion: Obesity/insulin resistance has a detrimental impact on vascular function. Reduced NO bioavailability and increased ROS generation from uncoupled eNOS and imbalanced release of COX products from COX-2 play a critical role in the development of these vascular alterations (C) 2010 Elsevier B.V. All rights reserved.

Effects of adolescent exposure to cocaine on locomotor activity and extracellular dopamine and glutamate levels in nucleus accumbens of DBA/2J mice

Adolescents differ from adults in their acute sensitivity to several drugs of abuse, but little is known about the long-term neurobehavioral effects of adolescent drug exposure. To explore this further, we evaluated the locomotor responses to repeated cocaine administration in adolescent and adult male DBA/2J mice and alterations in extracellular levels of dopamine (DA) and glutamate (GLU) in the nucleus accumbens (NAc) in response to a subsequent cocaine challenge. Adolescent and adult mice were treated daily with saline or cocaine (10 mg/kg, i.p) for 9 consecutive days. Ten days following the last injection, animals were implanted with microdialysis probes and 24 h later microdialysis samples were collected before and after an acute cocaine challenge. Adolescents but not adults demonstrated development of behavioral sensitization to cocaine. Microdialysis procedures revealed that cocaine-treated mice displayed greater peak increases in extracellular DA in response to a subsequent cocaine challenge as compared to saline-treated mice, in contrast with lower peak increases in extracellular GLU. While adults exhibited greater peaks in extracellular DA in response to cocaine than adolescents did, adolescent mice presented a more rapid onset of peak extracellular DA levels than adults. Our results indicate differences in the behavioral and neurochemical responses to cocaine in adolescent versus adult mice, which may be relevant to the increased risk of developing addiction in humans who are exposed to drugs of abuse during adolescence. (C) 2007 Elsevier B.V. All rights reserved.

Preliminary report: Leucine supplementation enhances glutamate dehydrogenase expression and restores glucose-induced insulin secretion in protein-malnourished rats

Low-protein diet impairs insulin secretion in response to nutrients and may induce several metabolic disorders including diabetes, obesity, and cardiovascular disease. In the present study, the influence of leucine supplementation on glutamate dehydrogenase (GDH) expression and glucose-induced insulin secretion (GIIS) was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal-protein diet (17%) without or with leucine supplementation or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine (1.5%) was supplied in the drinking water. Western blotting analysis revealed reduced GIN! expression in LP, whereas LPL displayed improved GDH expression, similar to control. The GHS and leucinc-induced insulin release were also enhanced in LPL compared with LP and similar to those observed in rats fed a normal-protein diet without leucine supplementation. In addition, GDH allosteric activators produced an increased insulin secretion in LPL. These findings indicate that leucine supplementation was able to increase GDH expression leading to Cl IS restoration, probably by improved leucine metabolic pathways. (C) 2010 Elsevier Inc. All rights reserved.

Insulin secretion in monosodium glutamate (MSG) obese rats submitted to aerobic exercise training

The present study was designed to evaluate the effects of aerobic exercise training on glucose tolerance and insulin secretion of obese male Wistar rats (monosodium glutamate [MSG] administration, 4mg/g-body weight, each other day, from birth to the 14th day). Fourteen weeks after the drug administration, the rats were separated into two groups: MSG-S (sedentary) and MSG-T (T = swimming, 1 h/day, 5 days/week, with an overload of 5% body weight for 10 weeks). Rats of the same age and strain injected with saline were used as control (C) and subdivided into two groups: C-S and C-T. Insulin and glucose responses during an oral glucose tolerance test (GTT) were evaluated by the estimation of the total areas under serum insulin (AI) and glucose (AG) curves. Glucose-induced insulin secretion by isolated pancreatic islets was also evaluated. MSG-S rats showed higher AI than C-rats while MSG-T rats presented lower AI than MSG-S rats. No differences in AG were observed among the 4 groups. Pancreatic islets from MSG-rats showed higher insulin secretion in response to low (2.8) and moderate (8.3 mM) concentrations of glucose than those from their control counterparts and no differences were observed between MSG-S and MSG-T rats. These results provide evidences that the hyperinsulinemia at low or moderate glucose concentrations observed in MSG-obese rats is, at least in part, a consequence of direct hypersecretion of the B cells and that chronic aerobic exercise is able to partially counteract the hyperinsulinemic state of these animals without disrupting glucose homeostasis.

Involvement of N-methyl-d-aspartate glutamate receptor and nitric oxide in cardiovascular responses to dynamic exercise in rats

Dynamic exercise evokes sustained cardiovascular responses, which are characterized by arterial pressure and heart rate increases. Although it is well accepted that there is central nervous system mediation of cardiovascular adjustments during exercise, information on the role of neural pathways and signaling mechanisms is limited. It has been reported that glutamate, by acting on NMDA receptors, evokes the release of nitric oxide through activation of neuronal nitric oxide synthase (nNOS) in the brain. In the present study, we tested the hypothesis that NMDA receptors and nNOS are involved in cardiovascular responses evoked by an acute bout of exercise on a rodent treadmill. Moreover, we investigated possible central sites mediating control of responses to exercise through the NMDA receptor-nitric oxide pathway. Intraperitoneal administration of the selective NMDA glutamate receptor antagonist dizocilpine maleate (MK-801) reduced both the arterial pressure and heart rate increase evoked by dynamic exercise. Intraperitoneal treatment with the preferential nNOS inhibitor 7-nitroindazole reduced exercise-evoked tachycardiac response without affecting the pressor response. Moreover, treadmill running increased NO formation in the medial prefrontal cortex (MPFC), bed nucleus of the stria teminalis (BNST) and periaqueductal gray (PAG), and this effect was inhibited by systemic pretreatment with MK-801. Our findings demonstrate that NMDA receptors and nNOS mediate the tachycardiac response to dynamic exercise, possibly through an NMDA receptor-NO signaling mechanism. However, NMDA receptors, but not nNOS, mediate the exercise-evoked pressor response. The present results also provide evidence that MPFC, BNST and PAG may modulate physiological adjustments during dynamic exercise through NMDA receptor-NO signaling. © 2013 Elsevier B.V.

GABA and glutamate transporters: new events and function in the vertebrate retina

The neural retina is a highly complex tissue composed of excitatory and inhibitory neurons and glial cells. Glutamate, the main excitatory neurotransmitter, mediates information transfer from photoreceptors, bipolar cells, and ganglion cells, whereas interneurons, mainly amacrine and horizontal cells, use γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter. In this review we place an emphasis on glutamate and GABA transporters as highly regulated molecules that play fundamental roles in neurotransmitter clearance, neurotransmitter release, and oxidative stress. We pharmacologically characterized glutamate transporters in chicken retina cells and identified two glutamate transporters: one Na+-dependent transporter and one Na+-independent transporter. The Na+-dependent uptake system presented characteristics related to the high-affinity xAG- system (EAAT1), and the Na+-independent uptake system presented characteristics related to the xCG- system, which highly contributes to glutamate transport in the retina. Glutamate shares the xCG- system with another amino acid, L-cysteine, suggesting the possible involvement of glutathione. Both transporter proteins are present mainly in Müller glial cells. GABA transporters (GATs) mediate high-affinity GABA uptake from the extracellular space and terminate the synaptic action of GABA in the central nervous system. GABA transporters can be modulated by molecules that act on specific sites to promote transporter phosphorylation and dephosphorylation. In addition to a role in the clearance of GABA, GATs may also release GABA through a reverse transport mechanism. In the chicken retina, a GAT-1 blocker, but not GAT2/3 blocker, was shown to inhibit GABA uptake, suggesting that GABA release from retina cells is mainly mediated by a GAT-1-like transporter.

Zooplankton oxygen consumption and nutrient release in relation to species composition, animals size and enviromental conditions in the Baltic Sea during May and August

[EN] Zooplankton metabolism in terms of oxygen consumption and ñutrient reléase (ammonia, phosphate) were measiu'ed in the Baltic Sea, a températe área with high envirormiental changes both in space and in time. Plankton of the surface layer were analysed with balance measurements in 4 size classes between 50 and 1000 nm during spring in 1988, 1990 and 1991, in summer 19^8 and 1990 as well. The use of electrón transport system (ETS), and the Glutamate Dehydrogenase (GDH) activity as indicators for respiration and ammonia reléase respectively, enlarged the data density and made a three dimensional resolution available (May 1990, 1991). Data are in the range of the latitudinal dependend magnitude. They reflect slight interannual, more seasonal and regional aspects. Animáis size, temperature, food concentration, and species composition influence the specific rates

Extracellular glutamate-GABA balance in the developing neocortex

It has been shown in the study that glutamate transporters (EAAT) are capable to modulate GABA transports (GAT). Here we also report that DL-TBOA, a non-transportable glutamate uptake blocker, eliminates GAT-mediated GABA release, while D-aspartate, an EAAT substrate, does not block the latter. The strength or even the operating mode of GABA uptake/release could be influenced by the work of EAATs. Considering the interaction between EAATs and GATs we can conclude that ambient glutamate and GABA levels are mutually dependent. The EAAT-GAT crosstalk observed in this work is mediated by EAAT1 and GAT-2/3. Since both transporters are Na+ dependent and mainly glial, next we investigated the role of [Na+]i in astrocytic-mediated glutamate uptake. We tested whether [Na+]i changes affect paired-pulse plasticity of STCs recorded from cortical layer 2/3 astrocytes. We report that an elevation of [Na+]i induced either by using a high [Na+]i intrapipette solution or by application of GABA slows STCs kinetics and decrease paired-pulse facilitation (PPF) of STCs at short inter-stimulus intervals. Moreover, GAT inhibitors decrease PPF of STCs under control conditions, suggesting that endogenous GABA operating via GATs influences EAAT-mediated transport

Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in Aplysia.

An increase in transmitter release accompanying long-term sensitization and facilitation occurs at the glutamatergic sensorimotor synapse of Aplysia. We report that a long-term increase in neuronal Glu uptake also accompanies long-term sensitization. Synaptosomes from pleural-pedal ganglia exhibited sodium-dependent, high-affinity Glu transport. Different treatments that induce long-term enhancement of the siphon-withdrawal reflex, or long-term synaptic facilitation increased Glu uptake. Moreover, 5-hydroxytryptamine, a treatment that induces long-term facilitation, also produced a long-term increase in Glu uptake in cultures of sensory neurons. The mechanism for the increase in uptake is an increase in the V(max) of transport. The long-term increase in Glu uptake appeared to be dependent on mRNA and protein synthesis, and transport through the Golgi, because 5,6-dichlorobenzimidazole riboside, emetine, and brefeldin A inhibited the increase in Glu uptake. Also, injection of emetine and 5,6-dichlorobenzimidazole into Aplysia prevented long-term sensitization. Synthesis of Glu itself may be regulated during long-term sensitization because the same treatments that produced an increase in Glu uptake also produced a parallel increase in Gln uptake. These results suggest that coordinated regulation of a number of different processes may be required to establish or maintain long-term synaptic facilitation.

Mechanisms for the release of dopamine from turtle retina

The retinal circuitry underlying the release of dopamine was examined in the turtle, Pseudemys scripta elegans, using neurochemical release studies, anatomical techniques, and biochemistry. There was a dose- and calcium-dependent release of dopamine from turtle retinas incubated in $\sp3$H-dopamine after perfusion of the GABA antagonist bicuculline. This indicated that dopamine release was tonically inhibited by GABA. Other putative retinal transmitters were examined. Glutamate antagonists selective for hyperpolarizing bipolar cells, such as 2,3-piperidine dicarboxylic acid (PDA), caused dose- and calcium-dependent release of dopamine from the retina. In contrast, release was not observed after perfusion with 4-aminophosphonobutyric acid, a specific antagonist of depolarizing bipolar cells. This indicated that depolarizing bipolar cells were not involved in retinal circuitry underlying the release of dopamine in the turtle retina. The release produced by PDA was blocked by bicuculline, indicating a polysynaptic mechanism of release. None of the other agents tested, which included carbachol, strychnine, dopamine uptake inhibitors, serotonin, tryptamine, muscimol, melatonin, or dopamine itself produced release.^ The cells capable of the release of dopamine were identified using both uptake autoradiography and immunocytochemical localization with dopamine antisera. The simplest circuitry based on these findings is signal transmission from photoreceptors to hyperpolarizing bipolar cells then to GABAergic cells, and finally to dopaminergic amacrine cells. ^

Characterization of neurotransmitter inputs to cholinergic amacrine cells of the rabbit retina: Analysis of ACh release

The cholinergic amacrine cells of the rabbit retinal are the only neurons which accumulate choline and also synthesize acetylcholine (ACh). It is widely accepted that the physiologically evoked release of acetylcholine can be taken as a measure of the activity of the entire cholinergic population. Initially, we examined the possibility that these cells receive excitatory input via glutamate receptors from glutamatergic neurons. Glutamate analogs were found to cause massive ACh release from the rabbit retina. Glutamate was found to activate several different receptor subtypes. Selective glutamate antagonists were used to separate the responses evoked by the different glutamate receptor subtypes. The kainate receptor was determined pharmacologically to be the subtype activated physiologically. Since bipolar cells make direct contact with cholinergic amacrine cells, our results support the hypothesis the bipolar cell neurotransmitter is glutamate. Although NMDA receptors can be activated by NMDA analogs, they are not activated during the physiologically evoked release of ACh. A separate study examined the possibility that L-homocysteate could be the bipolar cell neurotransmitter and the results placed serious constraints on this possibility.^ GABA$\sb{\rm A}$ agonists and antagonists are known to have powerful effects on ACh release from the rabbit retina. By pharmacologically blocking the excitatory input from bipolar cells, we attempted to determine the site of GABA$\sb{\rm A}$ input. Our results suggest that the predominant site of GABA$\sb{\rm A}$ input is onto the bipolar cells presynaptic to cholinergic amacrine cells. In a separate study, we found SR-95531 to be a potent and selective GABA$\sb{\rm A}$ receptor antagonist. In addition, GABA$\sb{\rm B}$ agonists and antagonists were found to have minor or no effects on ACh release. Glycine was also examined, its inhibitory effects were found to be very similar to GABA$\sb{\rm A}$ agonists. In contrast, strychnine was found to increase basal but inhibit light evoked ACh release. Additional results indicated that the predominant site of glycinergic input is onto the presynaptic bipolar cells. Our results suggest a different role for glycine compared to GABA in shaping the light evoked release of ACh from the rabbit retina. ^

Glutamate iontophoresis induces long-term potentiation in the absence of evoked presynaptic activity

$\rm\underline{L}$ong-$\rm\underline{t}$erm $\rm\underline{p}$otentiation (LTP) is a candidate cellular mechanism underlying mammalian learning and memory. Protocols that induce LTP typically involve afferent stimulation. The experiments described in this dissertation tested the hypothesis that LTP induction does not require presynaptic activity. The significance of this hypothesis is underscored by results suggesting that LTP expression may involve activity-dependent presynaptic changes. An induction protocol using glutamate iontophoresis was developed that reliably induces LTP in hippocampal slices without afferent stimulation (ionto-LTP). Ionto-LTP is induced when excitatory postsynaptic potentials are completely blocked with adenosine and $\rm\underline{t}$etrodo$\rm\underline{t}$o$\rm\underline{x}$in (TTX). These results suggest constraints on the involvement of presynaptic mechanisms and putative retrograde messengers in LTP induction and expression; namely, these processes must function without many forms of activity-dependent presynaptic processes.^ In testing the role of pre-and postsynaptic mechanisms in LTP expression whole-cell recordings were used to examine the frequency and amplitude of $\rm\underline{s}$pontaneous $\rm\underline{e}$xcitatory $\rm\underline{p}$o$\rm\underline{s}$ynaptic $\rm\underline{c}$urrents (sEPSCs) in CA1 pyramidal neurons. sEPSCs where comprised of an equal mixture of TTX insensitive miniature EPSCs and sEPSCs that appeared to result from spontaneous action potentials (i.e., TTX sensitive EPSCs). The detection of all sEPSCs was virtually eliminated by CNQX, suggesting that sEPSCs were glutamate mediated synaptic events. Changes in the amplitude and frequency sEPSCs were examined during the expression of ionto-LTP to obtain new information about the cellular location of mechanisms involved in synaptic plasticity. The findings of this dissertation show that ionto-LTP expression results from increased sEPSC amplitude in the absence of lasting increases in sEPSC frequency. Potentiation of sEPSC amplitude without changes in sEPSC frequency has been previously interpreted to be due to postsynaptic mechanisms. Although this interpretation is supported by findings from peripheral synapses, its application to the central nervous system is unclear. Therefore, alternative mechanisms are also considered in this dissertation. Models based on increased release probability for action potential dependent transmitter release appear insufficient to explain our results. The most straightforward interpretation of the results in this dissertation is that LTP induced by glutamate iontophoresis on dendrites of CA1 pyramidal neurons is mediated by postsynaptic mechanisms. ^

Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins, so-called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O-benzylated L-threo-β-hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy.

R-type Ca2+ currents evoke transmitter release at a rat central synapse

Voltage-dependent Ca2+ currents evoke synaptic transmitter release. Of six types of Ca2+ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca2+ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca2+ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca2+ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca2+ influx during a presynaptic action potential. This Ca2+ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca2+ currents. Activation of metabotropic glutamate receptors and γ-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca2+ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.

An alternative pathway for rod signals in the rodent retina: Rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors

In the mammalian retina, extensive processing of spatiotemporal and chromatic information occurs. One key principle in signal transfer through the retina is parallel processing. Two of these parallel pathways are the ON- and OFF-channels transmitting light and dark signals. This dual system is created in the outer plexiform layer, the first relay station in retinal signal transfer. Photoreceptors release glutamate onto ON- and OFF-type bipolar cells, which are functionally distinguished by their postsynaptic expression of different types of glutamate receptors, namely ionotropic and metabotropic glutamate receptors. In the current concept, rod photoreceptors connect only to rod bipolar cells (ON-type) and cone photoreceptors connect only to cone bipolar cells (ON- and OFF-type). We have studied the distribution of (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunits at the synapses in the outer plexiform layer of the rodent retina by immunoelectron microscopy and serial section reconstruction. We report a non-classical synaptic contact and an alternative pathway for rod signals in the retina. Rod photoreceptors made synaptic contact with putative OFF-cone bipolar cells that expressed the AMPA glutamate receptor subunits GluR1 and GluR2 on their dendrites. Thus, in the retina of mouse and rat, an alternative pathway for rod signals exists, where rod photoreceptors bypass the rod bipolar cell and directly excite OFF-cone bipolar cells through an ionotropic sign-conserving AMPA glutamate receptor.