RESUMEN
In stroke, the sudden deprivation of oxygen to neurons triggers a profuse release of glutamate that induces anoxic depolarization (AD) and leads to rapid cell death. Importantly, the latency of the glutamate-driven AD event largely dictates subsequent tissue damage. Although the contribution of synaptic glutamate during ischaemia is well-studied, the role of tonic (ambient) glutamate has received far less scrutiny. The majority of tonic, non-synaptic glutamate in the brain is governed by the cystine/glutamate antiporter, system xc - . Employing hippocampal slice electrophysiology, we showed that transgenic mice lacking a functional system xc - display longer latencies to AD and altered depolarizing waves compared to wild-type mice after total oxygen deprivation. Experiments which pharmacologically inhibited system xc - , as well as those manipulating tonic glutamate levels and those antagonizing glutamate receptors, revealed that the antiporter's putative effect on ambient glutamate precipitates the ischaemic cascade. As such, the current study yields novel insight into the pathogenesis of acute stroke and may direct future therapeutic interventions. KEY POINTS: Ischaemic stroke remains the leading cause of adult disability in the world, but efforts to reduce stroke severity have been plagued by failed translational attempts to mitigate glutamate excitotoxicity. Elucidating the ischaemic cascade, which within minutes leads to irreversible tissue damage induced by anoxic depolarization, must be a principal focus. Data presented here show that tonic, extrasynaptic glutamate supplied by system xc - synergizes with ischaemia-induced synaptic glutamate release to propagate AD and exacerbate depolarizing waves. Exploiting the role of system xc - and its obligate release of ambient glutamate could, therefore, be a novel therapeutic direction to attenuate the deleterious effects of acute stroke.
Asunto(s)
Isquemia Encefálica , Accidente Cerebrovascular , Ratones , Animales , Ácido Glutámico/metabolismo , Antiportadores/metabolismo , Isquemia , Ratones Transgénicos , Hipoxia , Hipocampo/metabolismo , Oxígeno/metabolismoRESUMEN
Most extracellular glutamate in the brain is released by xCT, a glial antiporter that exports glutamate and imports cystine. The function of xCT, and extracellular glutamate in general, remains unclear. Several lines of evidence suggest that glutamate from xCT could act in a paracrine fashion to suppress glutamatergic synapse strength by triggering removal of postsynaptic glutamate receptors. To test this idea, we used whole-cell patch-clamp electrophysiology and immunohistochemistry to quantify receptor number and synapse function in xCT knock-out mouse hippocampal CA3-CA1 synapses. Consistent with the hypothesis that xCT suppresses glutamate receptor number and synapse strength, xCT knock-out synapses showed increased AMPA receptor abundance with concomitant large enhancements of spontaneous and evoked synaptic transmission. We saw no evidence for changes in GABA receptor abundance or the overall number of glutamatergic synapses. The xCT knock-out phenotype was replicated by incubating slices in the xCT inhibitor (S)-4-carboxyphenylglycine, and consistent with the idea that xCT works by regulating extracellular glutamate, the xCT knock-out phenotype could be reproduced in controls by incubating the slices in glutamate-free aCSF. We conclude that glutamate secreted via xCT suppresses glutamatergic synapse strength by triggering removal of postsynaptic AMPA receptors.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/fisiología , Hipocampo/fisiología , Neuroglía/fisiología , Sinapsis/fisiología , Animales , Potenciales Postsinápticos Excitadores/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de ÓrganosRESUMEN
Synaptic vesicle secretion requires the assembly of fusogenic SNARE complexes. Consequently proteins that regulate SNARE complex formation can significantly impact synaptic strength. The SNARE binding protein tomosyn has been shown to potently inhibit exocytosis by sequestering SNARE proteins in nonfusogenic complexes. The tomosyn-SNARE interaction is regulated by protein kinase A (PKA), an enzyme implicated in learning and memory, suggesting tomosyn could be an important effector in PKA-dependent synaptic plasticity. We tested this hypothesis in Drosophila, in which the role of the PKA pathway in associative learning has been well established. We first determined that panneuronal tomosyn knockdown by RNAi enhanced synaptic strength at the Drosophila larval neuromuscular junction, by increasing the evoked response duration. We next assayed memory performance 3 min (early memory) and 3 h (late memory) after aversive olfactory learning. Whereas early memory was unaffected by tomosyn knockdown, late memory was reduced by 50%. Late memory is a composite of stable and labile components. Further analysis determined that tomosyn was specifically required for the anesthesia-sensitive, labile component, previously shown to require cAMP signaling via PKA in mushroom bodies. Together these data indicate that tomosyn has a conserved role in the regulation of synaptic transmission and provide behavioral evidence that tomosyn is involved in a specific component of late associative memory.
Asunto(s)
Memoria , Odorantes , Proteínas R-SNARE/fisiología , Transmisión Sináptica/fisiología , Animales , Drosophila/fisiología , Inmunohistoquímica , Cuerpos Pedunculados/fisiología , Unión Neuromuscular/fisiología , Proteínas R-SNARE/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The vesicle protein synaptotagmin I is the Ca(2+) sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca(2+) binding by the C(2)B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca(2+) binding results in vesicle fusion remains controversial. Ca(2+)-dependent interactions between synaptotagmin and SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment receptor) complexes and/or anionic membranes are possible effector interactions. However, no effector-interaction mutations to date impact synaptic transmission as severely as mutation of the C(2)B Ca(2+)-binding motif, suggesting that these interactions are facilitatory rather than essential. Here we use Drosophila to show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca(2+)-binding pockets of synaptotagmin. Mutation of this residue in the C(2)A domain (F286) resulted in a â¼50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C(2)B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that in syt(null) mutants, which is more severe than the phenotype of C(2)B Ca(2+)-binding mutants. Thus, mutation of a single, C(2)B hydrophobic residue required for Ca(2+)-dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin function in vivo to date. Our results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C(2)B domain of synaptotagmin, is the critical effector interaction for coupling Ca(2+) binding with vesicle fusion.
Asunto(s)
Calcio/metabolismo , Fusión de Membrana/fisiología , Vesículas Sinápticas/fisiología , Sinaptotagminas/metabolismo , Factores de Edad , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Drosophila , Proteínas de Drosophila/genética , Electrofisiología , Embrión no Mamífero , Potenciales Postsinápticos Excitadores/genética , Fraccionamiento de Campo-Flujo/métodos , Técnicas In Vitro , Fusión de Membrana/genética , Mutagénesis Sitio-Dirigida/métodos , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/fisiología , Estructura Terciaria de Proteína/genética , Ratas , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Alineación de Secuencia , Análisis Espectral , Sinaptotagminas/química , Sinaptotagminas/genéticaRESUMEN
The fruit fly (Drosophila melanogaster) is an extensively used and powerful, genetic model organism. However, chemical studies using individual flies have been limited by the animal's small size. Introduced here is a method to sample nanoliter hemolymph volumes from individual adult fruit-flies for chemical analysis. The technique results in an ability to distinguish hemolymph chemical variations with developmental stage, fly sex, and sampling conditions. Also presented is the means for two-point monitoring of hemolymph composition for individual flies.
Asunto(s)
Electroforesis Capilar , Hemolinfa/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Aminoácidos/química , Animales , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Femenino , Larva/química , Masculino , Nanotecnología , Factores SexualesRESUMEN
CASK ('calcium/calmodulin-dependent serine protein kinase'), also known in Drosophila as 'Caki' or 'Camguk/CMG', and in C. elegans as 'Lin-2', is thought to play an important role in cell-cell junction formation and at synapses in particular. To understand the role of CASK in synapse formation and function, we functionally and morphologically analyzed Drosophila embryonic and larval glutamatergic neuromuscular junctions (NMJs) after pan-cellular and tissue-specific manipulation of CASK expression. Our results show that Drosophila CASK is associated with both pre and postsynaptic membranes. Loss of presynaptic CASK led to less evoked synaptic transmission, fewer spontaneous synaptic events, and reduced synaptic vesicle cycling. These changes were accompanied by a reduction in the number of synapses but no change in overall NMJ size. Loss of postsynaptic CASK, on the other hand, caused reduced spontaneous synaptic current amplitudes and smaller glutamate-gated currents. These changes were accompanied by loss of postsynaptic glutamate receptors, but the receptor loss was subtype-specific: Only receptors containing GluRIIA subunits were lost in CASK mutants. Receptors containing GluRIIB were unaffected.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Guanilato-Quinasas/metabolismo , Animales , Proteínas de Drosophila/genética , Guanilato-Quinasas/genética , Unión Neuromuscular/metabolismo , Técnicas de Placa-Clamp , Densidad Postsináptica/metabolismo , Terminales Presinápticos/metabolismo , Interferencia de ARN , Receptores de Glutamato/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Mate choice is an evolutionarily critical decision that requires the detection of multiple sex-specific signals followed by central integration of these signals to direct appropriate behavior. The mechanisms controlling mate choice remain poorly understood. Here, we show that the glial amino-acid transporter genderblind controls whether Drosophila melanogaster males will attempt to mate with other males. Genderblind (gb) mutant males showed no alteration in heterosexual courtship or copulation, but were attracted to normally unappealing male species-specific chemosensory cues. As a result, genderblind mutant males courted and attempted to copulate with other Drosophila males. This homosexual behavior could be induced within hours using inducible RNAi, suggesting that genderblind controls nervous system function rather than its development. Consistent with this, and indicating that glial genderblind regulates ambient extracellular glutamate to suppress glutamatergic synapse strength in vivo, homosexual behavior could be turned on and off by altering glutamatergic transmission pharmacologically and/or genetically.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Cortejo , Proteínas de Drosophila/metabolismo , Neuroglía/metabolismo , Sinapsis/fisiología , Sistema de Transporte de Aminoácidos y+/genética , Animales , Animales Modificados Genéticamente , Conducta Animal , Sistema Nervioso Central/citología , Drosophila/fisiología , Proteínas de Drosophila/genética , Femenino , Ácido Glutámico/metabolismo , Homosexualidad/efectos de los fármacos , Masculino , Mutación/fisiología , ARN Interferente Pequeño/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/genéticaRESUMEN
Glia regulate brain physiology primarily by regulating the movement and concentration of substances in the extracellular fluid. Therefore, one approach to understanding the role of glia in brain physiology is to study what happens when glial transporters are removed or modified. The largest and most highly conserved class of transporter is solute carrier (SLC) proteins. SLC proteins are highly expressed in brain, and many are found in glia. The function of many SLC proteins in the brain--particularly in glia--is very poorly understood. SLC proteins can be relatively easily knocked out or modified in genetic model organisms to better understand glial function. Drosophila are popular genetic model organisms that offer a nice balance between genetic malleability and brain complexity. They are ideal for such an endeavor. This article lists and discusses SLC transporter family members that are expressed in both mouse and Drosophila glia, in an effort to provide a foundation for studies of glial SLC transporters using Drosophila as a model.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Neuroglía/metabolismo , Animales , Química Encefálica/genética , Química Encefálica/fisiología , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Ratones , Modelos GenéticosRESUMEN
The processes controlling glutamate receptor expression early in synaptogenesis are poorly understood. Here, we examine glutamate receptor (GluR) subunit mRNA expression and localization in Drosophila embryonic/larval neuromuscular junctions (NMJs). We show that postsynaptic GluR subunit gene expression is triggered by contact from the presynaptic nerve, approximately halfway through embryogenesis. After contact, GluRIIA and GluRIIB mRNA abundance rises quickly approximately 20-fold, then falls within a few hours back to very low levels. Protein abundance, however, gradually increases throughout development. At the same time that mRNA levels decrease following their initial spike, GluRIIA, GluRIIB, and GluRIIC subunit mRNA aggregates become visible in the cytoplasm of postsynaptic muscle cells. These mRNA aggregates do not colocalize with eIF4E, but nevertheless presumably represent mRNP particles of unknown function. Multiplex FISH shows that different GluR subunit mRNAs are found in different mRNPs. GluRIIC mRNPs are most common, followed by GluRIIA and then GluRIIB mRNPs. GluR mRNP density is not increased near NMJs, for any subunit; if anything, GluR mRNP density is highest away from NMJs and near nuclei. These results reveal some of the earliest events in postsynaptic development and provide a foundation for future studies of GluR mRNA biology.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster , ARN Mensajero/biosíntesis , Receptores de Glutamato/biosíntesis , Ribonucleoproteínas/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Hibridación Fluorescente in Situ , Larva/genética , Larva/metabolismo , Unión Neuromuscular/embriología , Unión Neuromuscular/metabolismo , Subunidades de Proteína/metabolismo , Receptores de Glutamato/genética , Ribonucleoproteínas/genéticaRESUMEN
Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.
Asunto(s)
Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Animales , Drosophila , Inmunohistoquímica , Microscopía Electrónica , Mutación , Técnicas de Placa-Clamp , Sinapsis/fisiología , Sinapsis/ultraestructura , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructura , Proteínas de Transporte Vesicular de Glutamato/genéticaRESUMEN
This study investigated the effect of different sampling environments on hemolymph amino acid content of individual Drosophila melanogaster larvae. Hemolymph was collected from individual third instar larvae under cold-anesthetized, awake, and stress conditions. Qualitative and quantitative hemolymph amino acid analyses were performed via capillary electrophoresis with laser-induced fluorescence detection. The hemolymph amino acid concentrations, particularly arginine, glutamate, and taurine, changed significantly depending on the prior-to-sample-collection environments. Hemolymph amino acid analyses of six different Drosophila genotypes including two control genotypes and four mutant alleles were also carried out. Two mutant genotypes with over and under expression of a putative cystine-glutamate exchanger subunit were significantly different from each other with respect to their hemolymph glutamate, glycine, lysine, and taurine levels. Hemolymph amino acid analyses of stressed larvae of two control and two mutant genotypes indicated that behavior-related hemolymph chemical changes are also genotype dependent.
Asunto(s)
Aminoácidos/sangre , Conducta Animal/fisiología , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Variación Genética , Hemolinfa/química , Estrés Fisiológico , Alelos , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos/genética , Anestesia , Animales , Proteínas de Drosophila/genética , Electroforesis Capilar , Genotipo , Larva , Mutación , Estrés Fisiológico/genéticaRESUMEN
Measurements suggest that the hemolymph glutamate concentrations in Drosophila are relatively high. This raises the possibility that extracellular glutamate could be an important regulator of glutamatergic transmission in vivo. Using voltage clamp electrophysiology, we found that synaptic currents in D. melanogaster larval neuromuscular junctions are reduced by extracellular glutamate (EC50: approximately 0.4 mM), such that only 10-30% of receptors were functionally available in 1 mM extracellular glutamate. The kinetics of synaptic currents were also slowed in a dose-dependent fashion (EC50: approximately 1 mM), consistent with the idea that extracellular glutamate preferentially removes the fastest-desensitizing receptors from the functional pool. Prolonged exposure (several hours) to extracellular glutamate also triggers loss of glutamate receptor immunoreactivity from neuromuscular junctions. To determine whether this receptor loss requires that glutamate bind directly to the lost receptors, we examined glutamate-dependent loss of receptor immunoreactivity in larvae with glutamate receptor ligand binding mutations. Our results suggest that glutamate-dependent receptor loss requires binding of glutamate directly to the lost receptors. To determine whether lost receptor protein is degraded or merely redistributed, we used immunoblots. Results suggest that glutamate receptor protein is redistributed, but not degraded, after prolonged exposure to high extracellular glutamate.
Asunto(s)
Drosophila/metabolismo , Ácido Glutámico/farmacología , Unión Neuromuscular/efectos de los fármacos , Receptores de Glutamato/metabolismo , Animales , Animales Modificados Genéticamente , Biofisica , Relación Dosis-Respuesta a Droga , Proteínas de Drosophila/genética , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Líquido Extracelular/efectos de los fármacos , Larva , Mutación/fisiología , Unión Neuromuscular/fisiología , Técnicas de Placa-Clamp/métodos , Transporte de Proteínas/efectos de los fármacos , Factores de TiempoRESUMEN
We hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, we identified and functionally characterized a novel Drosophila xCT gene, which we subsequently named "genderblind" (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination of gb causes a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering, gb mutants show a large (200-300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additional in situ pharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, our results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamate in vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitized in vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Neuroglía/metabolismo , Receptores de Glutamato/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Neurotransmisores/metabolismo , Vesículas Sinápticas/metabolismo , Distribución TisularRESUMEN
Many neuroscientists assume that ambient extracellular glutamate concentrations in the nervous system are biologically negligible under nonpathological conditions. This assumption is false. Hundreds of studies over several decades suggest that ambient extracellular glutamate levels in the intact mammalian brain are approximately 0.5 to approximately 5 microM. This has important implications. Glutamate receptors are desensitized by glutamate concentrations significantly lower than needed for receptor activation; 0.5 to 5 microM of glutamate is high enough to cause constitutive desensitization of most glutamate receptors. Therefore, most glutamate receptors in vivo may be constitutively desensitized, and ambient extracellular glutamate and receptor desensitization may be potent but generally unrecognized regulators of synaptic transmission. Unfortunately, the mechanisms regulating ambient extracellular glutamate and glutamate receptor desensitization remain poorly understood and understudied.
Asunto(s)
Líquido Extracelular/metabolismo , Ácido Glutámico/metabolismo , Transmisión Sináptica/fisiología , Animales , Encéfalo/metabolismo , Encéfalo/fisiología , Ácido Glutámico/farmacología , Transmisión Sináptica/efectos de los fármacosRESUMEN
We hypothesized that presynaptic glutamate regulates postsynaptic ionotropic glutamate receptor number during synaptogenesis. To test this idea, we genetically manipulated presynaptic glutamate levels at the glutamatergic Drosophila neuromuscular junction (NMJ), then microscopically and electrophysiologically measured postsynaptic glutamate receptor field size and function. Our data show that presynaptic glutamate is a strong negative regulator of postsynaptic receptor field size and function during development. Glutamate-triggered receptor downregulation was not affected by block of synaptic vesicle fusion, demonstrating that receptors are regulated by nonvesicular glutamate release. Our results reveal an elegant mechanism for receptor field regulation during synaptogenesis and reveal a nonpathological role for nonvesicular glutamate release at the synapse.
Asunto(s)
Envejecimiento/metabolismo , Ácido Glutámico/fisiología , Receptores de Glutamato/metabolismo , Animales , Regulación hacia Abajo/fisiología , Drosophila/embriología , Electrofisiología , Embrión no Mamífero/metabolismo , Unión Neuromuscular/embriología , Terminales Presinápticos/metabolismo , Receptores de Glutamato/fisiología , Sinapsis/metabolismo , Distribución Tisular/fisiologíaRESUMEN
The Drosophila gene c12.2 was isolated in a screen examining mRNA binding proteins. Drosophila c12.2 is the mouse Vwa8 homolog. Various genome-wide associated studies have linked human Vwa8 to both neurological and oncological pathologies, which include autism, bipolar disorder, comorbid migraine, and acute myeloid leukemia, however, the function and role of the VWA8 protein remain poorly understood. To further analyze the Vwa8 gene in mouse, gene structure, protein homology modeling, and gene expression patterns were examined throughout mouse development. Our analyses indicate that the mouse Vwa8 gene produces two transcripts; the full-length Vwa8a is highly expressed relative to the truncated Vwa8b transcript across all developmental time points and tissues analyzed. Protein homology modeling indicates that VWA8a belongs to a novel protein superfamily containing both the midasin and cytoplasmic dynein 1 heavy chain 1 proteins. These data establish the development timeline and expression profile for both Vwa8a and Vwa8b, paving the way for future studies to determine the cellular role(s) of this highly conserved protein family.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Adenosina Trifosfatasas/genética , Animales , Embrión de Mamíferos/citología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Filogenia , Isoformas de Proteínas , Análisis Espacio-TemporalRESUMEN
This work demonstrates a reduced tip µ-low-flow-push-pull perfusion technique for ex vivo sampling of the extracellular space of mouse hippocampal brain slices. Concentric fused-silica capillary probes are pulled by an in-house gravity puller with a butane flame producing probe tips averaging an overall outer diameter of 30.3 ± 8 µm. The 10-30 nL/min perfusion flow rate through the probe generates an average recovery of 90%. Sampling was performed with mouse brain tissue slices to characterize basal neurotransmitter content in this model system. Samples were collected from hippocampal tissue slices at a volume of 200 nL per sample. Sample arginine, histamine, lysine, glycine, glutamate, and aspartate content was quantified by micellar electrokinetic chromatography with LED-induced fluorescence detection. Primary amine content was sampled over several hours to determine evidence for tissue damage and loss of extracellular content from the tissue slice. Overall, all amino acid concentrations trended lower as an effect of time relative to tissue slicing. There were significant concentration decreases seen for histamine, lysine, and aspartate between time points 0-2 and 2-6 h (p < 0.05) relative to tissue slicing. Analysis of averaged sampling experiments does not appear to reveal significant probe-insertion-related amino acid changes. The work presented shows the applicability of an 80% reduction of probe tip size relative to previous designs for the collection of extracellular content from thin tissue slices.
Asunto(s)
Espacio Extracelular/metabolismo , Hipocampo/metabolismo , Neurotransmisores/metabolismo , Técnicas de Cultivo de Tejidos/instrumentación , Aminoácidos/metabolismo , Animales , Calibración , Cromatografía/instrumentación , Diseño de Equipo , Masculino , Ratones , Microscopía Electrónica de Rastreo , Dióxido de SilicioRESUMEN
Glutamatergic Drosophila neuromuscular junctions contain two spatially, biophysically, and pharmacologically distinct subtypes of postsynaptic glutamate receptor (GluR). These receptor subtypes appear to be molecularly identical except that A receptors contain the subunit GluRIIA (but not GluRIIB), and B receptors contain the subunit GluRIIB (but not GluRIIA). A- and B-type receptors are coexpressed in the same cells, in which they form homotypic clusters. During development, A- and B-type receptors can be differentially regulated. The mechanisms that allow differential segregation and regulation of A- and B-type receptors are unknown. Presumably, A- and B-type receptors are differentially anchored to the membrane cytoskeleton, but essentially nothing is known about how Drosophila glutamate receptors are localized or anchored. We identified coracle, a homolog of mammalian brain 4.1 proteins, in yeast two-hybrid and genetic screens for proteins that interact with and localize Drosophila glutamate receptors. Coracle interacts with the C terminus of GluRIIA but not GluRIIB. To test whether coracle is required for glutamate receptor localization, we immunocytochemically and electrophysiologically examined receptors in coracle mutants. In coracle mutants, synaptic A-type receptors are lost, but there is no detectable change in B-type receptor function or localization. Pharmacological disruption of postsynaptic actin phenocopies the coracle mutants, suggesting that A-type receptors are anchored to the actin cytoskeleton via coracle, whereas B-type receptors are anchored at the synapse by another (yet unknown) mechanism.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Proteínas de la Membrana/fisiología , Receptores de Glutamato/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Factores Despolimerizantes de la Actina/farmacología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Larva , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Unión Neuromuscular/embriología , Unión Neuromuscular/metabolismo , Técnicas de Placa-Clamp , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Receptores de Glutamato/química , Tiazoles/farmacología , Tiazolidinas , Técnicas del Sistema de Dos HíbridosRESUMEN
A Drosophila forward genetic screen for mutants with defective synaptic development identified bad reception (brec). Homozygous brec mutants are embryonic lethal, paralyzed, and show no detectable synaptic transmission at the glutamatergic neuromuscular junction (NMJ). Genetic mapping, complementation tests, and genomic sequencing show that brec mutations disrupt a previously uncharacterized ionotropic glutamate receptor subunit, named here "GluRIID." GluRIID is expressed in the postsynaptic domain of the NMJ, as well as widely throughout the synaptic neuropil of the CNS. In the NMJ of null brec mutants, all known glutamate receptor subunits are undetectable by immunocytochemistry, and all functional glutamate receptors are eliminated. Thus, we conclude that GluRIID is essential for the assembly and/or stabilization of glutamate receptors in the NMJ. In null brec mutant embryos, the frequency of periodic excitatory currents in motor neurons is significantly reduced, demonstrating that CNS motor pattern activity is regulated by GluRIID. Although synaptic development and molecular differentiation appear otherwise unperturbed in null mutants, viable hypomorphic brec mutants display dramatically undergrown NMJs by the end of larval development, suggesting that GluRIID-dependent central pattern activity regulates peripheral synaptic growth. These studies reveal GluRIID as a newly identified glutamate receptor subunit that is essential for glutamate receptor assembly/stabilization in the peripheral NMJ and required for properly patterned motor output in the CNS.
Asunto(s)
Proteínas de Drosophila/fisiología , Unión Neuromuscular/fisiología , Neurópilo/metabolismo , Receptores de Glutamato/fisiología , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/metabolismo , Estimulación Eléctrica/métodos , Embrión no Mamífero , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Excitadores/efectos de la radiación , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica/métodos , Microscopía Confocal/métodos , Biología Molecular/métodos , Técnicas de Placa-Clamp/métodos , Subunidades de Proteína/deficiencia , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Glutamato/deficiencia , Proteínas Supresoras de Tumor/metabolismo , Proteína Letal Asociada a bcl/deficienciaRESUMEN
BACKGROUND: Drosophila discs-large (DLG) is the sole representative of a large class of mammalian MAGUKs, including human DLG, SAP 97, SAP102, and PSD-95. MAGUKs are thought to be critical for postsynaptic assembly at glutamatergic synapses. However, glutamate receptor cluster formation has never been examined in Drosophila DLG mutants. The fly neuromuscular junction (NMJ) is a genetically-malleable model glutamatergic synapse widely used to address questions regarding the molecular mechanisms of synapse formation and growth. Here, we use immunohistochemistry, confocal microscopy, and electrophysiology to examine whether fly NMJ glutamate receptor clusters form normally in DLG mutants. We also address the question of how DLG itself is localized to the synapse by testing whether presynaptic innervation is required for postsynaptic DLG clustering, and whether DLG localization requires the presence of postsynaptic glutamate receptors. RESULTS: There are thought to be two classes of glutamate receptors in the Drosophila NMJ: 1) receptors that contain the subunit GluRIIA, and 2) receptors that contain the subunit GluRIIB. In DLG mutants, antibody staining for the glutamate receptor subunit GluRIIA is normal, but antibody staining for the glutamate receptor subunit GluRIIB is significantly reduced. Electrophysiological analysis shows an overall loss of functional postsynaptic glutamate receptors, along with changes in receptor biophysical properties that are consistent with a selective loss of GluRIIB from the synapse. In uninnervated postsynaptic muscles, neither glutamate receptors nor DLG cluster at synapses. DLG clusters normally in the complete absence of glutamate receptors. CONCLUSIONS: Our results suggest that DLG controls glutamate receptor subunit composition by selectively stabilizing GluRIIB-containing receptors at the synapse. We also show that DLG, like glutamate receptors, is localized only after the presynaptic neuron contacts the postsynaptic cell. We hypothesize that glutamate receptors and DLG cluster in response to parallel signals from the presynaptic neuron, after which DLG regulates subunit composition by stabilizing (probably indirectly) receptors that contain the GluRIIB subunit. The mechanism(s) stabilizing GluRIIA-containing receptors remains unknown.