A genetic analysis of glutamaterigic function in Drosophila

Neurotransmitters are essential for communication between neurons and hence are vital in the overall integrative functioning of the nervous system. Previous work on acetylcholine metabolism in the fruit fly, Drosophila melanogaster, has also raised the possibility that transmitter metabolism may play a prominent role in either the achievement or maintenance of the normal structure of the central nervous system in this species. Unfortunately, acetylcholine is rather poorly characterized as a neurotransmitter in Drosophila; consequently, we have begun an analysis of the role of glutamate (probably the best characterized transmitter in this organism) in the formation and/or maintenance of nervous system structure. We present here the results of a series of preliminary analyses. (1) To suggest where glutamatergic function may be localized, an examination of the spatial distribution of high affinity [³H]-glutamate binding sites are presented. (2) We present the results of an analysis of the spatial and temporal distribution of enzymatic activities thought to be important in the regulation of transmitter-glutamate pools (i.e., glutamate oxaloacetic transaminase, glutaminase, and glutamate dehydrogenase). (3) To begin to examine whether mutations in any of these functions are capable of affecting glutamaterigic activity, we present the results of an initial genetic analysis of one enzymatic function, glutamate oxaloacetic transaminase (GOT), chosen because of its differential distribution within the adult central nervous system and musculature.     

On the role of normal acetylcholine metabolism in the formation and maintenance of the Drosophila nervous system

We have examined the requirement for normal acetylcholine metabolism in the formation and maintenance of the larval and adult central nervous system in Drosophila melanogaster. By using mutations at the Ace and Cha loci, which respectively encode the degradative and synthetic enzymes for acetylcholine (ACh), acetylcholinesterase (AChE), and choline acetyltransferase (ChAT), we have been able to disrupt acetylcholine metabolism in situ. An ultrastructural analysis of embryonic nervous tissue lacking either enzymatic function has indicated that while neither function is required for the formation of the larval central nervous system, each is required for the subsequent maintenance of its structural integrity and function. Using temperature sensitive mutations at the Cha locus, the normal developmental profile of ChAT activity during the late larval and pupal stages was disrupted. Subsequent examination of the morphology and behavior of the treated animals has indicated that normal acetylcholine metabolism is not required for the initial formation of the adult nervous system, but is required for the subsequent maintenance of its structural integrity and function. The results obtained in these studies are discussed with respect to data presented on the adult distribution of the cholinergic markers' AChE activity and ChAT immunoreactivity. The projections of adult peripheral neurons innervating Ace+ tissue from Ace cuticular clones has been examined to address the nature of the structure of Ace neuropil. Normal projections are apparently achieved and maintained, suggesting that the defects seen in adult Ace mosaics arise as an aberrant intracellular organization of morphologically normal cells.     

From glutamate co-release to vesicular synergy: vesicular glutamate transporters

Recent data indicate that 'classical' neurotransmitters can also act as co-transmitters. This notion has been strengthened by the demonstration that three vesicular glutamate transporters (vesicular glutamate transporter 1 (VGLUT1), VGLUT2 and VGLUT3) are present in central monoamine, acetylcholine and GABA neurons, as well as in primarily glutamatergic neurons. Thus, intriguing questions are raised about the morphological and functional organization of neuronal systems endowed with such a dual signalling capacity. In addition to glutamate co-release, vesicular synergy - a process leading to enhanced packaging of the 'primary' transmitter - is increasingly recognized as a major property of the glutamatergic co-phenotype. The behavioural relevance of this co-phenotype is presently the focus of considerable interest.     

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

Summary Using a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.     

Cloning and functional identification of a neuronal glutamine transporter

Glutamine is the preferred precursor for the neurotransmitter pool of glutamate, the major excitatory transmitter in the mammalian central nervous system. We have isolated a complementary DNA clone (designated GlnT) encoding a plasma membrane glutamine transporter from glutamatergic neurons in culture, and its properties have been examined using the T7 vaccinia system in fibroblasts. When GlnT is transfected into CV-1 cells, L-glutamine is the preferred substrate. Transport is Na(+)-dependent and inhibited by alpha-methylaminoisobutyric acid, a specific inhibitor of neutral amino acid transport system A. Kinetic analysis of glutamine uptake by GlnT is saturable, with a Michaelis constant (K(m)) of 489 +/- 88 microM at pH 7.4. Glutamine uptake mediated by GlnT is pH-sensitive with a 5-fold greater efficiency of uptake at pH 8.2 than at pH 6.6. Only the maximal velocity of transport increases without a significant change in K(m). The distribution of GlnT mRNA and protein in the central nervous system is widespread and is expressed on neurons that use glutamate as their neurotransmitter. In cultured cerebellar granule cells, GlnT is expressed only on neurons and is absent from astrocytes. GlnT expression increases concomitantly with the morphologic and functional differentiation of these cells in vitro, consistent with its role of supplying glutamatergic neurons with their neurotransmitter precursor. GlnT is the first member of the system A family of neutral amino acid transporters with 11 putative membrane-spanning domains and is a potential target to modulate presynaptic glutamatergic function.     

Importance of astrocytic inactivation of synaptically released glutamate for cell survival in the central nervous system--are astrocytes vulnerable to low intracellular glutamate concentrations?

Multiple levels of neuron-astrocyte interactions do exist at glutamatergic synapses, glial glutamate transporters being involved in most of them. Inactivation of synaptically released glutamate is not only important for the phasic aspect of glutamatergic transmission but also for astrocyte metabolism, which supply neurons with different metabolic precursors, and for cell survival in the central nervous system. Alteration of glutamate transport, which leads to abnormally high extracellular glutamate levels, has been involved in numerous neurodegenerative diseases. There are different ways by which elevated extracellular levels of glutamate can be toxic. Excitotoxic mechanisms, involving overstimulation of glutamate receptors, have been shown to induce the death of neurons and oligodendrocytes, but not of astrocytes. Oxidative glutamate toxicity, which can affect every cell type of the central nervous system, is currently viewed as the consequence of altered cystine transport, leading in turn to reduced glutathione synthesis and oxidative stress. This review summarizes the functional implications of astroglial glutamate transport and the consequences of its alteration. Emphasis is laid on our recent finding that alteration of glutamate transport, by depleting intracellular stores of glutamate, can induce oxidative toxicity in astrocytes. The consequences for the other cell types of the central nervous system are discussed in terms of neuron dependency on astrocytes for glutathione synthesis and therefore oxidative stress protection.     

Regional and subcellular distribution of cysteine sulfinate transaminase in rat nervous system

The distribution of the cysteine sulfinate transaminase activity in adult and newborn rat central nervous system was studied and compared with the distribution of the glutamate oxaloacetate transaminase activity. The subcellular localization of both enzyme activities was also investigated. These experiments suggest that both enzymes, sometimes considered as identical, are different.     

Aspartate Aminotransferase and Glutamate Dehydrogenase Activities in the Squid Giant Nerve

Abstract: The present study sought to investigate the presence and distribution of some enzymatic activities involved in the metabolism of glutamate in the giant nerve fiber of the tropical squid Sepioteuthis sepioidea . Specific activities of aspartate aminotransferase and glutamate dehydrogenase were evaluated in homogenates of the isolated giant fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. The activities of both enzymes were present in the tissue. The specific activity of aspartate aminotransferase was similar in axoplasm and sheaths. However, the specific activity of glutamate dehydrogenase was an order of magnitude higher in the sheaths. This finding is discussed in the framework of the hypothesis that proposes that a differential distribution of the enzymes of the glutamatergic system between the axonal and neuroglial compartments forms part of a system of communication between these cells whose neuronal signal may be glutamate.     

Identification of an endogenous glutamatergic transmitter system controlling excitability and conductivity of atrial cardiomyocytes

As an excitatory transmitter system, the glutamatergic transmitter system controls excitability and conductivity of neurons. Since both cardiomyocytes and neurons are excitable cells, we hypothesized that cardiomyocytes may also be regulated by a similar system. Here, we have demonstrated that atrial cardiomyocytes have an intrinsic glutamatergic transmitter system, which regulates the generation and propagation of action potentials. First, there are abundant vesicles containing glutamate beneath the plasma membrane of rat atrial cardiomyocytes. Second, rat atrial cardiomyocytes express key elements of the glutamatergic transmitter system, such as the glutamate metabolic enzyme, ionotropic glutamate receptors (iGluRs), and glutamate transporters. Third, iGluR agonists evoke iGluR-gated currents and decrease the threshold of electrical excitability in rat atrial cardiomyocytes. Fourth, iGluR antagonists strikingly attenuate the conduction velocity of electrical impulses in rat atrial myocardium both in vitro and in vivo. Knockdown of GRIA3 or GRIN1, two highly expressed iGluR subtypes in atria, drastically decreased the excitatory firing rate and slowed down the electrical conduction velocity in cultured human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocyte monolayers. Finally, iGluR antagonists effectively prevent and terminate atrial fibrillation in a rat isolated heart model. In addition, the key elements of the glutamatergic transmitter system are also present and show electrophysiological functions in human atrial cardiomyocytes. In conclusion, our data reveal an intrinsic glutamatergic transmitter system directly modulating excitability and conductivity of atrial cardiomyocytes through controlling iGluR-gated currents. Manipulation of this system may open potential new avenues for therapeutic intervention of cardiac arrhythmias.Subject terms: Cell biology, Molecular biology     

Redefining chemical anatomy of glutamatergic neurotransmission

With the recent identification of the two isoforms of vesicular glutamate transporters VGLUT1 and VGLUT2 and of the presumed neuronal glutamine transporter SAT1 novel tools have been made available to unequivocally define the anatomy of glutamatergic pathways on the cellular and synaptic level. Using highly specific antisera and cRNA probes two distinct glutamatergic pathways expressing either VGLUT1 or VGLUT2 could be detected throughout the central nervous system. Areas where VGLUT1 predominated included the cerebral and cerebellar cortex and the hippocampus. VGLUT2 was mainly expressed in the thalamus, hypothalamus and brain stem. VGLUT1 and VGLUT2 synapses exhibited distinct region- and pathway-specific relationships with each other and with other classical transmitter and peptidergic systems. The glutamine transporter SAT1 was expressed in CNS neurons and in ependymal cells. Neuronal SAT1 expression comprised virtually all glutamatergic neurons but also specific subsets of cholinergic, GABAergic and aminergic neurons in the CNS. In addition to widespread expression of VGLUT1 and VGLUT2 in the CNS, peripheral tissues such as sensory neurons and pancreatic islet cells differentially expressed VGLUT isoforms and SAT1.
Our results suggest pathway-specific functional duality in the regulation of vesicular glutamate release at excitatory synapses and provide evidence for glutamine transport and metabolism in excitatory glutamatergic and diverse nonglutamatergic neurons as well.     

Beyond the role of glutamate as a neurotransmitter

Glutamate is the principal excitatory neurotransmitter of the central nervous system, but many studies have expanded its functional repertoire by showing that glutamate receptors are present in a variety of non-excitable cells. How does glutamate receptor activation modulate their activity? Do non-excitable cells release glutamate, and, if so, how? These questions remain enigmatic. Here, we review the current knowledge on glutamatergic signalling in non-neuronal cells, with a special emphasis on astrocytes.     

Glutamate, a neurotransmitter--and so much more. A synopsis of Wierzba III

It appears almost incredible that the first indications that glutamate excites brain tissue were obtained during the second half of the 20th century, that vesicles containing glutamate were demonstrated in glutamatergic neurons less than 25 years ago, and that glutamate was not accepted as the major excitatory transmitter until about the same time. During this span of time it has also become realized that glutamate is so much more than a conventional neurotransmitter: (1) astrocytes express vesicles accumulating glutamate by vesicular transporters akin to the vesicular glutamate transporters in glutamatergic neurons, and they release glutamate by exocytosis; (2) a series of metabolic processes in astrocytes (glutamate uptake, glutamine synthetase activity, glutamine release) are involved in neuronal reutilization of transmitter glutamate; (3) glutamine may also be utilized for synthesis of GABA, the major inhibitory transmitter; (4) de novo synthesis of glutamate accounts for 20% of cerebral glucose metabolism, all of which initially occurs in astrocytes, and at steady state a corresponding amount of glutamate is oxidatively degraded, mainly or exclusively in astrocytes; (5) tissue contents of glutamate/glutamine increase during enhanced glutamatergic activity, i.e., astrocytic de novo synthesis exceeds astrocytic metabolic degradation of glutamate.     

ATP-Dependent Glutamate Uptake into Synaptic Vesicles from Cerebellar Mutant Mice

The ATP-dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP-dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP-dependent glutamate uptake system is reduced by 68% in the staggerer and 57-67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP-dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.     

Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice.

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron-neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin-deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin-deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age-matched wild-type neurons during the first 3 weeks in culture. These results demonstrate that neuron-specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction.     

Biochemical Characterization of the Yeast Yarrowia lipolytica Overproducing Carboxylic Acids from Ethanol: Nitrogen Metabolism Enzymes

A comparative assay of nitrogen metabolism enzymes in the Yarrowia lipolytica mutant N1 grown under conditions promoting the overproduction of either -ketoglutaric acid (KGA) or citric acid showed that the overproduction of KGA correlates with an increase in the activities of the NAD- and NADP-linked glutamate dehydrogenase, glutamic–pyruvic transaminase, and glutamic–oxaloacetic transaminase reactions. These reactions are likely to be responsible for the overproduction of KGA by this mutant. In contrast, the overproduction of citric acid correlated with a decline in the activities of the NAD- and NADP-linked glutamate dehydrogenases and with an increase in the activities of glutamine synthetase and glutamate synthase.     

Nociceptin/orphanin FQ and neurotransmitter release in the central nervous system

In this article, the effect of nociceptin (orphanin FQ) on transmitter release in the central nervous system in vitro and in vivo is reviewed. Nociceptin inhibits the electrically or K(+)-evoked noradrenaline, dopamine, serotonin, and glutamate release in brain slices from guinea-pig, rat, and mouse. This effect is usually naloxone-resistant but antagonized by OP(4) receptor antagonists like [Phe(1)psi(CH(2)-NH)Gly(2)]-nociceptin(1-13)NH(2). In the rat in vivo, nociceptin diminishes acetylcholine release in the striatum, reduces dopamine release, and prevents the stimulatory effect of morphine on this transmitter in the nucleus accumbens and also elevates extracellular glutamate and gamma-aminobutyric acid levels in mesencephalic dopaminergic areas. The effect of nociceptin on the mesencephalic dopaminergic system might explain its actions on motor behavior.     

Some components of the cholinergic system in the prawn Palaemonetes varians (Leach)

1. An enzyme similar to mammalian acetylcholinesterase is found in high activity in the nervous tissue of Palaemonetes varians, i.e. eyes plus stalks, brain, suboesophageal ganglion and ventral cord. Acetylcholinesterase is also found associated with the abdominal muscles. Multiple enzyme forms are found in extracts of nervous tissues and muscles by electrophoresis and isoelectric focusing. 2. Cholinesterase is present in high activity in the stomatogastric system of P. varians. Three electrophoretically separable forms are found, having isoelectric points at pH4.2, 4.5 and 5.4. 3. Approx. 50% of the total acetylcholinesterase activity, approx. 80% of the choline acetyltransferase activity and 100% of the acetylcholine equivalents are found associated with the nervous tissue. Among the tissues examined, eyes plus stalks were the richest source of both choline acetyltransferase and acetylcholine equivalents. Suboesophageal ganglion and brain also contained large amounts of these components. 4. The distribution of these components could support the function of acetylcholine as a central and/or sensory transmitter in P. varians.     

Localization of glutamate-like immunoreactive neurons in the central and peripheral nervous system of the adult and developing pond snail, Lymnaea stagnalis

We investigated the distribution and projection patterns of central and peripheral glutamate-like immunoreactive (GLU-LIR) neurons in the adult and developing nervous system of Lymnaea. Altogether, 50-60 GLU-LIR neurons are present in the adult central nervous system. GLU-LIR labeling is shown in the interganglionic bundle system and at the varicosities in neuropil of the central ganglia. In the periphery, the foot, lip, and tentacle contain numerous GLU-LIR bipolar sensory neurons. In the juvenile Lymnaea, GLU-LIR elements at the periphery display a pattern of distribution similar to that seen in adults, whereas labeled neurons increase in number in the different ganglia of the central nervous system from juvenile stage P1 up to adulthood. During embryogenesis, GLU-LIR innervation can be detected first at the 50% stage of embryonic development (the E50% stage) in the neuropil of the cerebral and pedal ganglia, followed by the emergence of labeled pedal nerve roots at the E75% stage. Before hatching, at the E90% stage, a few GLU-LIR sensory cells can be found in the caudal foot region. Our findings indicate a wide range of occurrence and a broad role for glutamate in the gastropod nervous system; hence they provide a basis for future studies on glutamatergic events in networks underlying different behaviors.     

Development of the glutamate system in rabbit retina

We have investigated two characteristics of the glutamate system in the developing rabbit retina. 1) Glutamate immunoreactivity was observed at birth within developing processes of four cell types; two of which, photoreceptors and ganglion cells, are known to be glutamatergic in the adult. Two other cell types, type A horizontal cells and amacrine cells, are immunoreactive to both glutamate and GABA at birth, suggesting that endogenous pools of glutamate in GABAergic neurons serve as precursor for GABA synthesis. Thus it appears that endogenous glutamate pools are present within neurons prior to synaptogenesis as part of the early expression of either the glutamate or GABA transmitter phenotype. 2) Analysis of³H-glutamate metabolism during retinal development showed that rapid conversion of glutamate to glutamine does not occur until the second postnatal week, coincident with the expression of Muller (glial) cell activity. In the absence of glial metabolism in the neonate, extracellular concentrations of glutamate remain relatively high and are likely to have major effects on neuronal maturation.Special issue dedicated to Dr. Frederick E. Samson