NPY receptor subtype specification for behavioral adaptive strategies during limited food access.

The neuropeptide Y (NPY) system in the brain regulates a wide variety of behavioral, metabolic and hormonal homeostatic processes required for energy balance control. During times of limited food availability, NPY promotes behavioral hyperactivity necessary to explore and prepare for novel food resources. As NPY can act via 5 different receptor subtypes, we investigated the path through which NPY affects different behavioral components relevant for adaptation to such conditions. We tested NPY Y1 and Y2 receptor knockout mice and their wild-type littermate controls in a daily scheduled limited food access paradigm with unlimited access to running wheel. Here we show that NPY Y1 receptor deficient mice lack the expression of appetitive behavior and that NPY Y2 receptors control the level of hyperactive behavior under these conditions. Thus, receptor specificity determines the differential expression of NPY-mediated behavioral adaptations to overcome a negative energy status.

Increased glutamine synthetase but normal EAAT2 expression in platelets of ALS patients.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease and glutamate excitotoxicity has been implicated in its pathogenesis. Platelets contain a glutamate uptake system and express components of the glutamate-glutamine cycle, such as the predominant glial excitatory amino acid transporter 2 (EAAT2). In several neurological diseases platelets have proven to be systemic markers for the disease. We compared properties of key components of the glutamate-glutamine cycle in blood platelets of ALS patients and healthy controls. Platelets were analyzed for (3)H-glutamate uptake in the presence or absence of thrombin and for EAAT2 and glutamine synthetase protein expression by Western blotting. Platelets of ALS patients showed a 37% increase in expression of glutamine synthetase, but normal expression of glutamate transporter EAAT2. Glutamate uptake in resting or thrombin-stimulated platelets did not differ significantly between platelets from ALS patients and controls. Thrombin-stimulation resulted in about a seven-fold increase in glutamate uptake. Our data suggest that glutamine synthetase may be a peripheral marker of ALS and encourage further investigation into the role of this enzyme in ALS.

Thrombin-stimulated glutamate uptake in human platelets is predominantly mediated by the glial glutamate transporter EAAT2.

Glutamate toxicity has been implicated in the pathogenesis of various neurological diseases. Glial glutamate transporters play a key role in the regulation of extracellular glutamate levels in the brain by removing glutamate from the extracellular fluid. Since human blood platelets possess an active glutamate uptake system, they have been used as a peripheral model of glutamate transport in the central nervous system (CNS). The present study is aimed at identifying the glutamate transporter on blood platelets, and to asses the influence of platelet activation on glutamate uptake. Platelets from healthy donors showed Na+-dependent glutamate uptake (Km, 3.5+/-0.9 microM; Vmax, 2.8+/-0.2 pmol glutamate/75 x 10(6)platelets/30 min), which could be blocked dose-dependently by the EAAT specific inhibitors DL-threo-E-benzyloxyaspartate (TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and high concentrations of the EAAT2 inhibitor dihydrokainate (DHK). Analysis of platelet homogenates on Western blots showed EAAT2 as the predominant glutamate transporter. Platelet activation by thrombin caused an increase in glutamate uptake, which could be inhibited by TBOA and the EAAT2 inhibitor DHK. Kinetic analysis showed recruitment of new transporters to the membrane. Indeed, Western blot analysis of subcellular fractions revealed that alpha-granules, which fuse with the membrane upon thrombin stimulation, contained significant EAAT2 immunoreactivity. Inhibition of the second messengers involved in alpha-granule secretion (protein kinase C, phosphatidylinositol-3-kinase) inhibited thrombin-stimulated uptake, but not basal uptake. These data show that the glial EAAT2 is the predominant glutamate transporter on blood platelets and suggest, that thrombin increases glutamate uptake capacity by recruiting new transporters (EAAT2) from alpha-granules.

Synaptosomal glutamate and GABA transport in patients with temporal lobe epilepsy.

High-affinity glutamate and GABA transporters found in the plasma membrane of neurons and glial cells terminate neurotransmission by rapidly removing extracellular transmitter. Impairment of transporter function has been implicated in the pathophysiologic mechanisms underlying epileptogenesis. We characterized glutamate and gamma-aminobutyric acid (GABA) transport in synaptosomes, isolated from neocortical and hippocampal biopsies of patients with temporal lobe epilepsy (TLE). We analyzed K(+)-evoked release in the presence and absence of Ca(2+) to determine vesicular and transporter-mediated release, respectively. We also analyzed (3)H-glutamate and (3)H-GABA uptake, the effect of glutamate uptake inhibitors L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and DL-threo-beta-benzyloxyaspartate (TBOA), and GABA uptake inhibitor N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid (SK&F 89976-A). Neocortical synaptosomes from TLE patients did not show vesicular glutamate release, strongly reduced transporter-mediated release, and an increased basal release compared to that in rat synaptosomes. Furthermore, basal release was less sensitive to tPDC, and (3)H-glutamate uptake was reduced compared to that in rat synaptosomes. Vesicular GABA release from neocortical synaptosomes of TLE patients was reduced compared to that in rat synaptosomes, whereas transporter-mediated release was hardly affected. Furthermore, basal GABA release was more than doubled, but neither basal nor stimulated release were increased by SK&F 89976-A, which did significantly increase both types of GABA release in rat synaptosomes. Finally, (3)H-GABA uptake by synaptosomes from TLE patients was reduced significantly in hippocampus (0.19 +/- 0.04%), compared to that in neocortex (0.32 +/- 0.04%). Control experiments with human peritumoral cortical tissue suggest that impaired uptake of glutamate, but not of GABA, was caused in part by the hypoxic state of the biopsy. Our findings provide evidence for impaired function of glutamate and GABA transporters in human TLE.

Characterization of neocortical and hippocampal synaptosomes from temporal lobe epilepsy patients.

To investigate epilepsy-associated changes in the presynaptic terminal, we isolated and characterized synaptosomes from biopsies resected during surgical treatment of drug-resistant temporal lobe epilepsy (TLE) patients. Our main findings are: (1) The yield of synaptosomal protein from biopsies of epilepsy patients was about 25% of that from rat brain. Synaptosomal preparations were essentially free of glial contaminations. (2) Synaptosomes from TLE patients and naive rat brain, quickly responded to K(+)-depolarization with a 70% increase in intrasynaptosomal Ca(2+) ([Ca(2+)](i)), and a 40% increase in B-50/GAP-43 phosphorylation. (3) Neocortical and hippocampal synaptosomes from TLE patients contained 20-50% of the glutamate and gamma-aminobutyric acid (GABA) contents of rat cortical synaptosomes. (4) Although the absolute amount of glutamate and GABA released under basal conditions from neocortical synaptosomes of TLE patients was lower than from rat synaptosomes, basal release expressed as percentage of total content was higher (16.4% and 17.3%, respectively) than in rat (11.5% and 9. 9%, respectively). (5) Depolarization-induced glutamate and GABA release from neocortical synaptosomes from TLE patients was smaller than from rat synaptosomes (3.9% and 13.0% vs. 21.9% and 25.0%, respectively). (6) Analysis of breakdown of glial fibrillary acid protein (GFAP) indicates that resection time (anoxic period during the operation) is a critical parameter for the quality of the synaptosomes. We conclude that highly pure and viable synaptosomes can be isolated even from highly sclerotic human epileptic tissue. Our data show that in studies on human synaptosomes it is of critical importance to distinguish methodological (i.e., resection time) from pathology-related abnormalities.

Anti-B-50 (GAP-43) antibodies decrease exocytosis of glutamate in permeated synaptosomes.

The involvement of the protein kinase C substrate, B-50 (GAP-43), in the release of glutamate from small clear-cored vesicles in streptolysin-O-permeated synaptosomes was studied by using anti-B-50 antibodies. Glutamate release was induced from endogenous as well as 3H-labelled pools in a [Ca(2+)]-dependent manner. This Ca(2+)-induced release was partially ATP dependent and blocked by the light-chain fragment of tetanus toxin, demonstrating its vesicular nature. Comparison of the effects of anti-B-50 antibodies on glutamate and noradrenaline release from permeated synaptosomes revealed two major differences. Firstly, Ca(2+)-induced glutamate release was decreased only partially by anti-B-50 antibodies, whereas Ca(2+)-induced noradrenaline release was inhibited almost completely. Secondly, anti-B-50 antibodies significantly reduced basal glutamate release, but did not affect basal noradrenaline release. In view of the differences in exocytotic mechanisms of small clear-cored vesicles and large dense-cored vesicles, these data indicate that B-50 is important in the regulation of exocytosis of both types of neurotransmitters, probably at stages of vesicle recycling and/or vesicle recruitment, rather than in the Ca(2+)-induced fusion step.

4-Aminopyridine differentially affects the spontaneous release of radiolabelled transmitters from rat brain slices in vitro.

4-Aminopyridine increased the release of [3H]noradrenaline from dorsal hippocampus slices in vitro in a concentration-dependent manner. When the slices were exposed to 4-aminopyridine for 5 min, the overflow of radioactivity returned to pre-exposure values within 20-25 min. When the exposure of the slices was continued, a sustained enhancement of the release of [3H]noradrenaline was observed for the duration of the exposure. 4-Aminopyridine, 10(-4) M, had an effect of similar magnitude, or an even more pronounced effect, on the release of [3H]catecholamine from cortex, septum, periaqueductal gray and striatum slices. The effects of the compound on the release of [3H]5-hydroxytryptamine and [14C]acetylcholine were less pronounced. At this concentration 4-aminopyridine had no effect on the release of [3H]D-aspartate from hippocampus or septum slices, whereas the effect on the release of this transmitter in striatal slices was marginal. The effect of 4-aminopyridine on the release of [3H]noradrenaline in hippocampus slices was largely dependent on the presence of Ca2+ in the superfusion medium. This was also the case for the effect on the release of [3H]noradrenaline from preloaded dorsal hippocampus synaptosomes. In the presence of nitrendipine the effect of 4-aminopyridine was dose-dependently reduced, but the maximal reduction, at a nitrendipine concentration of 10(-4) M, was only 40%. Cd2+ completely abolished the effect of 4-aminopyridine on the release of [3H]noradrenaline. These results confirm that the enhancing effect of 4-aminopyridine on the release of [3H]noradrenaline depends on the entry of extracellular Ca2+ into the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)