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ROLES OF NMDA RECEPTOR AND EAAC1 TRANSPORTER IN MODULATION OF EXTRACELLULAR GLUTAMATE BY LOW AND HIGH AFFINITY AMPA RECEPTORS IN CEREBELLUM IN VIVO. DIFFERENTIAL ALTERATION IN CHRONIC HYPERAMMONEMIA Andrea Cabrera-Pastor, Lucas Taoro, Marta Llansola, and Vicente Felipo ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.5b00212 • Publication Date (Web): 02 Oct 2015 Downloaded from http://pubs.acs.org on October 5, 2015
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ACS Chemical Neuroscience
ROLES OF NMDA RECEPTOR AND EAAC1 TRANSPORTER IN MODULATION OF EXTRACELLULAR GLUTAMATE BY LOW AND HIGH AFFINITY AMPA RECEPTORS IN CEREBELLUM IN VIVO. DIFFERENTIAL ALTERATION IN CHRONIC HYPERAMMONEMIA.
Andrea Cabrera-Pastor, Lucas Taoro, Marta Llansola and Vicente Felipo*
Laboratory of Neurobiology, Centro de Investigación Príncipe Felipe, 46012, Valencia, Spain
* CORRESPONDENCE: Vicente Felipo, Laboratory of Neurobiology, Centro de Investigacion Principe Felipe, Calle Eduardo Primo Yufera, 3, 46012 Valencia. Spain e-mail:
[email protected] Tel: 34 963289680
Fax: 34 963289701
ABBREVIATIONS: AMPA: (RS)- -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; cGMP: cyclic guanosine mono-phosphate; GABA: -Aminobutyric acid; GluR: glutamate receptor subunit; mGluR: metabotropic glutamate receptor ; MK-801: (5S,10R)-(+)-5-Methyl-10,11-dihydro5H- dibenzo[a,d]cyclohepten-5,10-imine; NMDA: N-Methyl-Daspartic acid; NO: nitric oxide; EAAC1: Excitatory Amino Acid Carrier 1; GLAST: Glutamate/Aspartate transporter; GLT-1: glutamate transporter-1; HPLC: high performance liquid chromatography.
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ABSTRACT The roles of high- and low-affinity AMPA receptors in modulating extracellular glutamate in cerebellum remain unclear. Altered glutamatergic neurotransmission is involved in neurological alterations in hyperammonemia, which affects differently high- and low-affinity AMPA receptors. The aims were to assess by in vivo microdialysis: a) the effects of high- and lowaffinity AMPA receptors activation on extracellular glutamate in cerebellum; b) whether chronic hyperammonemia alters extracellular glutamate modulation by high- and/or low-affinity AMPA receptors; c) the contribution of NMDA receptors and EAAC1 transporter to AMPA-induced changes in extracellular glutamate. In control rats high affinity receptors activation does not affect extracellular glutamate, but increases glutamate if NMDA receptors are blocked. Low affinity AMPA receptors activation increases transiently extracellular glutamate followed by reduction below basal levels and return to basal values. The reduction is associated with transient increased membrane expression of EAAC1 and is prevented by blocking NMDA receptors. Blocking NMDA receptors with MK-801 induces a transient increase in extracellular glutamate which is associated with reduced membrane expression of EEAC1 followed by increased membrane expression of the glutamate transporter GLT-1. Chronic hyperammonemia does not affect responses to activation of low affinity AMPA receptors. Activation of high affinity AMPA receptors increases extracellular glutamate in hyperammonemic rats by an NMDA receptor-dependent mechanism. In conclusion, these results show that there is a tightly controlled interplay between AMPA and NMDA receptors and EAAC1 transporter in controlling extracellular glutamate. Hyperammonemia alters high- but not low-affinity AMPA receptors. Key words: AMPA receptors, NMDA receptors, extracellular glutamate, hyperammonemia, glutamate uptake, EAAC1 glutamate transporter
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INTRODUCTION Glutamate modulates cognitive and motor functions through activation of ionotropic (iGluRs) and metabotropic glutamate receptors. There are three main types of iGluRs: NMDA, AMPA and kainate receptors. AMPA receptors are composed of four types of subunits (GluR1-GluR4). Activation of AMPA receptors allows the entry of Na+. Receptors lacking GluR2 subunits are also permeable to Ca2+ (1,2). There are high- and low-affinity AMPA receptors in brain which seem to be inter-convertible states of the same receptors (3,4). Most AMPA receptors are of the low affinity type (4) and contain the GluR2 subunit (5), which prevents Ca2+transport. In agreement with this, Cabrera-Pastor et al (6) suggested that in cerebellum in vivo high affinity AMPA receptors transport Na+, K+ and Ca2+ while low affinity receptors transport mainly Na+ and K+ but not Ca2+. Moreover, Cabrera-Pastor et al (6) also showed that in cerebellum in vivo low AMPA concentrations (0.1 mM) are enough to nearly completely activate Ca2+-permeable AMPA receptors, which have higher affinity for AMPA than Ca2+-impermeable receptors (6). AMPA receptors activation may lead to NMDA receptors activation (6-8). Activation of NMDA or AMPA receptors increases extracellular glutamate (9-17). However, excessive or sustained activation of iGluRs is neurotoxic and extracellular glutamate must be rapidly reduced. Glutamate transporters terminate the excitatory signal by rapid glutamate uptake into cells. There are three main glutamate transporters GLAST and GLT-1 (astrocytic) and EAAC1 (neuronal). EAAC1 seems to play a main role in the rapid modulation of glutamatergic synaptic transmission (18,19). For example, Levenson et al (20) showed that induction of long-term potentiation in the CA1 region of hippocampus is associated with an NMDA receptor-dependent translocation of the EAAC1 glutamate transporter to the plasma membrane and increased uptake of glutamate. Waxman et al (19) reported a dual modulation of the glutamate transporter EAAC1 by NMDA receptors. Under basal conditions NMDA receptors enhance EAAC1 membrane expression, which is also enhanced by mild activation of NMDA receptors (i.e. with 10 µM NMDA). However, robust activation of NMDA receptors 3 ACS Paragon Plus Environment
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(i.e. with 100 µM NMDA) or AMPA receptors (i.e. with 100 µM AMPA + cyclothiazide) reduces membrane expression of EAAC1. To make compatible rapid neurotransmission with keeping properly low glutamate levels, there is a rapid interaction between iGluRs activation and surface expression of EAAC1 (19). The sequence of events would be: activation of NMDA and/or AMPA receptors, increased extracellular glutamate, increased EAAC1surface expression, increased glutamate uptake, rapid decrease of extracellular glutamate. This allows a transient increase of glutamate and a rapid return to low levels while maintaining proper glutamatergic neurotransmission. Altered glutamatergic neurotransmission is involved in cognitive and motor alterations in many pathological situations including chronic hyperammonemia and hepatic encephalopathy (21-26). Patients with chronic liver diseases have reduced capacity to eliminate ammonia in the liver and, as a consequence, show hyperammonemia which increases ammonia levels in brain and is a main contributor to the cognitive and motor alterations in patients with hepatic encephalopathy (27). Altered glutamatergic neurotransmission due to hyperammonemia contributes to the neurological alterations in hepatic encephalopathy (21,25). Clarifying the mechanisms by which hyperammonemia alters glutamatergic neurotransmission may identify targets to improve neurological function in hepatic encephalopathy (24). Hyperammonemia impairs the glutamate-nitric oxide (NO)-cGMP pathway in cerebellum in vivo, which is responsible for reduced ability to learn a Y maze task (28-30). The function of this pathway is modulated by different neurotransmitter receptors including GABAA (30) and high and low affinity AMPA receptors (31,6). Hyperammonemia affects differently high- and low-affinity AMPA receptors. Hyperammonemia reduces activation of Ca2+-permeable AMPA receptors but increases Na+ entering through high affinity AMPA receptors, resulting in NMDA receptors activation (6). The modulation of extracellular glutamate by AMPA receptors has not been studied in cerebellum in vivo even in normal rats. 4 ACS Paragon Plus Environment
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The aims of this work were to assess: a) the effects of activation of high- and low-affinity AMPA receptors on extracellular glutamate in cerebellum in vivo b) whether chronic hyperammonemia alters the modulation of extracellular glutamate by highand/or low-affinity AMPA receptors c) the contribution of NMDA receptors and EAAC1 transporter to AMPA-induced changes in extracellular glutamate
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RESULTS Time-course of the modulation of extracellular glutamate and of membrane expression of EAAC1 by activation of low affinity AMPA receptors. Activation of low affinity AMPA receptors was achieved by using 0.3 mM AMPA in the in vivo microdialysis studies and 2 mM AMPA in the studies on membrane expression of EAAC1 in cerebellar slices. In control rats administration of AMPA (0.3 mM) through the microdialysis probe induced a significant increase in extracellular glutamate, reaching 158-189% of basal in the two fractions after AMPA administration (fractions 5-6 in Fig. 1A). This increase was transient and was followed by a reduction of extracellular glutamate below the basal levels before AMPA administration. Glutamate levels were maintained at 47-50% of basal in fractions 8-11 (fractions 4-7 after AMPA) (Fig. 1A). After this period, extracellular glutamate returned to basal values in fractions 12-13 (Fig. 1A). Hyperammonemia did not affect the transient increase in glutamate or the subsequent decrease and normalization induced by 0.3 mM AMPA (Fig. 1A). The basal levels of extracellular glutamate were significantly (p
