RESUMO
Glia-neuron partnership is important for inner retinal homeostasis and any disturbances may result in retinal ganglion cell (RGC) death. Müller cells support RGCs with essential functions such as removing excess glutamate and providing energy sources. The aim was to explore the impact of Müller cells on RGC survival. To investigate the Müller cell/RGC interactions we developed a coculture model, in which primary Müller cells were grown in inserts on top of pure primary RGC cultures. The impact of starvation and mitochondrial inhibition on the Müller cell ability to protect RGCs was studied. Moreover, the ability of Müller cells to remove glutamate from the extracellular space was investigated. RGC survival was evaluated by cell viability assays and glutamate uptake was assessed by kinetic uptake assays. We demonstrated a significantly increased RGC survival in presence of untreated and prestarved Müller cells. Additionally, prestarved Müller cells significantly increased RGC survival after mitochondrial inhibition. Finally, we revealed a significantly increased ability to take up glutamate in starved Müller cells. Overall, our study confirms essential roles of Müller cells in RGC survival. We suggest that targeting Müller cell function could have potential for future treatment strategies to prevent blinding neurodegenerative retinal diseases.
Assuntos
Técnicas de Cocultura , Células Ependimogliais , Mitocôndrias/metabolismo , Células Ganglionares da Retina , Animais , Sobrevivência Celular , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Camundongos , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismoRESUMO
Schizophrenia patients are often obese or overweight and poor dietary choices appear to be a factor in this phenomenon. Poor diet has been found to have complex consequences for the mental state of patients. Thus, this study investigated whether an unhealthy diet [i.e. high fat diet (HFD)] impacts on the behaviour of a genetic mouse model for the schizophrenia risk gene neuregulin 1 (i.e. transmembrane domain Nrg1 mutant mice: Nrg1 HET). Female Nrg1 HET and wild-type-like littermates (WT) were fed with either HFD or a control chow diet. The mice were tested for baseline (e.g. anxiety) and schizophrenia-relevant behaviours after 7 weeks of diet exposure. HFD increased body weight and impaired glucose tolerance in all mice. Only Nrg1 females on HFD displayed a hyper-locomotive phenotype as locomotion-suppressive effects of HFD were only evident in WT mice. HFD also induced an anxiety-like response and increased freezing in the context and the cued version of the fear conditioning task. Importantly, CHOW-fed Nrg1 females displayed impaired social recognition memory, which was absent in HFD-fed mutants. Sensorimotor gating deficits of Nrg1 females were not affected by diet. In summary, HFD had complex effects on the behavioural phenotype of test mice and attenuated particular cognitive deficits of Nrg1 mutant females. This topic requires further investigations thereby also considering other dietary factors of relevance for schizophrenia as well as interactive effects of diet with medication and sex.
Assuntos
Dieta Hiperlipídica , Neuregulina-1/genética , Esquizofrenia/dietoterapia , Animais , Comportamento Animal/fisiologia , Peso Corporal/genética , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Feminino , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos , Atividade Motora/genética , Neuregulina-1/metabolismo , Reflexo de Sobressalto/genética , Fatores de Risco , Esquizofrenia/genética , Filtro Sensorial/genéticaRESUMO
About half of the human brain is white matter, characterized by axons covered in myelin, which facilitates the high speed of nerve signals from one brain area to another. At the time of myelination, the oligodendrocytes that synthesize myelin require a large amount of energy for this task. Conditions that deprive the tissue of energy can kill the oligodendrocytes. During brain development, the oligodendrocytes may use lactate as an alternative source of energy and material for myelin formation. Mature oligodendrocytes, however, can release lactate through the myelin sheath as nutrient for axons. In addition, lactate carries signals as a volume transmitter. Myelin thus seems to serve as a provider of substrates and signals for axons, and not as a mere insulator. We review the fluxes of lactate in white matter and their significance in brain function.
Assuntos
Axônios/metabolismo , Ácido Láctico/metabolismo , Bainha de Mielina/metabolismo , Substância Branca/metabolismo , Animais , Axônios/ultraestrutura , Humanos , Bainha de Mielina/fisiologia , Oligodendroglia/metabolismo , Transdução de SinaisRESUMO
A state of low dopaminergic activity has been implicated in attention-deficit/hyperactivity disorder (ADHD). The clinical symptoms of ADHD include inattention, impulsivity and hyperactivity, as well as impaired learning; dopaminergic modulation of the functions in the hippocampus is important to both learning and memory. To determine dopamine receptor (DR) density in a well-established animal model for ADHD, we quantified the dopamine D5 receptors in the hippocampus in the spontaneously hypertensive rat. We used immunofluorescence microscopy and immunogold electron microscopy to quantify the dopamine D5 receptor density on CA1 pyramidal cell somas and dendrites and dendritic spines in the stratum radiatum and stratum oriens. The density of the dopamine D5 receptors was significantly lower in the cytoplasm of pyramidal cell somas in the spontaneously hypertensive rat compared to the control, indicating a reduced reservoir for insertion of receptors into the plasma membrane. DRs are important for long-term potentiation and long-term depression, hence the deficit may contribute to the learning difficulties in individuals with the diagnosis of ADHD.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Região CA1 Hipocampal/metabolismo , Receptores de Dopamina D5/metabolismo , Animais , Dendritos/metabolismo , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Células Piramidais/metabolismo , RatosRESUMO
The Kavli Prize in Neuroscience was awarded for the third time in September 2012, by the Norwegian Academy of Science and Letters in Oslo. The accompanying Kavli Prize Symposium on Neuroscience, held in Bergen and Trondheim, was a showcase of excellence in neuroscience research. The common theme of the Symposium presentations was the mechanisms by which animals adapt to their environment. The symposium speakers--Michael Greenberg, Erin Schuman, Chiara Cirelli, Michael Meaney, Catherine Dulac, Hopi Hoekstra, and Stanislas Dehaene--covered topics ranging from the molecular and cellular levels to the systems level and behavior. Thus a single amino acid change in a transcriptional repressor can disrupt gene regulation through neural activity (Greenberg). Deep sequencing analysis of the neuropil transcriptome indicates that a large fraction of the synaptic proteome is synthesized in situ in axons and dendrites, permitting local regulation (Schuman). The nature of the 'reset' function that makes animals dependent of sleep is being revealed (Cirelli). Maternal behavior can cause changes in gene expression that stably modify behavior in the offspring (Meaney). Removal of a single sensory channel protein in the vomero-nasal organ can switch off male-specific and switch on female-specific innate behavior of mice in response to environmental stimulation (Dulac). Innate behaviors can be stably transmitted from parent to offspring through generations even when those behaviors cannot be expressed, as illustrated by the elaborate burrowing behavior in a rodent species, in which independent genetic regions regulate distinct modules of the burrowing pattern (Hoekstra). Finally, at the other extreme of the nature-nurture scale, functional magnetic resonance imaging (fMRI) analysis in children and adults identified a brain area specifically involved in reading (Dehaene). As the area must originally have developed for a purpose other than reading, such as shape recognition, this illustrates the use of a previously formed neural structure to tackle a new challenge.
Assuntos
Adaptação Psicológica/fisiologia , Distinções e Prêmios , Encéfalo/fisiologia , Meio Ambiente , Rede Nervosa/fisiologia , Comportamento Social , Animais , Humanos , NoruegaRESUMO
Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.
Assuntos
Astrócitos/metabolismo , Ácido Glutâmico/metabolismo , Ouro , Hipocampo/metabolismo , Imuno-Histoquímica/métodos , Serina/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Células Cultivadas , Ratos , Ratos WistarRESUMO
Functional studies indicate that the dopamine D5 receptor is involved in synaptic transmission in the hippocampus. However, previous anatomical studies have detected D5 receptor labelling primarily on the soma and main dendrites of CA1 pyramidal cells and on dendritic spines in monkey but not in rats. In order to get a better understanding of putative dopamine function in the hippocampus, we quantified the D5 receptor immunoreactivity on the pyramidal cell somas and on spines and dendrites in stratum radiatum and stratum oriens in the hippocampal CA1 region of rats by quantitative immunofluorescence and immunogold electron microscopy. The quantitative immunogold results revealed a higher labelling density on dendritic spines, notably at their synaptic membranes, compared to pyramidal cell somas and dendrites. Hence, dopamine could have effects on spines as well as on somas and dendrites. The labelling density was similar on spines in stratum oriens and stratum radiatum, but the presence of labelling varied between the spines within each stratum, indicating that the effect of dopamine could be diverse between different spines.
Assuntos
Química Encefálica , Região CA1 Hipocampal/química , Receptores de Dopamina D5/análise , Sinapses/química , Animais , Western Blotting , Região CA1 Hipocampal/metabolismo , Imunofluorescência , Microscopia Eletrônica de Transmissão , Ratos , Ratos Wistar , Receptores de Dopamina D5/metabolismo , Sinapses/metabolismoRESUMO
The Kavli Prizes were awarded for the first time in Oslo, Norway on September 9, 2008 to seven of the world's most prominent scientists in astrophysics, nanoscience and neuroscience. The astrophysics prize was awarded jointly to Maarten Schmidt, of the California Institute of Technology, USA, and Donald Lynden-Bell, of Cambridge University, UK; the nanoscience prize was awarded jointly to Louis E. Brus, of Columbia University, USA, and Sumio Iijima, of Meijo University, Japan; and the neuroscience prize was awarded jointly to Pasko Rakic, of the Yale University School of Medicine, USA, Thomas Jessell, of Columbia University, USA, and Sten Grillner, of the Karolinska Institute, Sweden. The Kavli Prize is a joint venture of the Kavli Foundation, the Norwegian Academy of Science and Letters, and the Norwegian Ministry of Education and Research. The Kavli Prize Inaugural Symposium on Neuroscience was held at the University of Oslo on 8 September, 2008, organized by L.H. Bergersen, E. Moser M.-B. Moser, and J. Storm-Mathisen. At this Symposium, seven leading neuroscientists described their groundbreaking work, which encompasses some of the most important recent advances in the field of neuroscience, from molecule to synapse to network to behavior. The Symposium was a fitting tribute to Fred Kavli's vision of neuroscience as an outstanding area of progress, and to the achievements of the winners of the first Kavli Prize in Neuroscience. The main points of the Symposium presentations are summarized below.
Assuntos
Memória/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Distinções e Prêmios , Encéfalo/fisiologia , Genes MHC Classe I/fisiologia , Hipocampo/fisiologia , Humanos , Aprendizagem/fisiologia , Vias Neurais/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Neurociências , Proteínas de Transporte de Neurotransmissores/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
The aims of this study were to investigate the sarcomeric accumulation and expression of heat shock proteins (HSPs) after two bouts of maximal eccentric exercise. Twenty-four subjects performed two bouts of 70 maximal voluntary eccentric actions using the elbow flexors in one arm. The bouts were separated by 3 wk. The changes in concentric (60 degrees/s) and isometric (90 degrees) force-generating capacity were monitored for 9 days after each bout, and biopsies were taken 1 and 48 h and 4 and 7 days after bout 1 and 1 and 48 h after bout 2. The content of HSP27, alphaB-crystallin, HSP70, and desmin in the cytosolic and cytoskeleton/myofibrillar fractions of homogenized muscle samples was determined by immunoassays, and the cellular and subcellular localization of the HSPs in the myofibrillar structure was analyzed by conventional and confocal immunofluorescence microscopy and quantitative electron microscopy. The force-generating capacity was reduced by approximately 50% and did not recover completely during the 3 wk following bout 1. After bout 2, the subjects recovered within 4 days. The HSP levels increased in the cytosolic fraction after bout 1, especially HSP70 (approximately 300% 2-7 days after exercise). Increased levels of HSP27, alphaB-crystallin, and HSP70 were found in the cytoskeletal/myofibrillar fraction after both bouts, despite reduced damage after bout 2. At the ultrastructural level, HSP27 and alphaB-crystallin accumulated in Z-disks, in intermediate desmin-like structures (alphaB-crystallin), and in areas of myofibrillar disruption. In conclusion, HSP27 and alphaB-crystallin accumulated in myofibrillar structures, especially in the Z-disks and the intermediate structures (desmin). The function of the small HSPs is possibly to stabilize and protect the myofibrillar structures during and after unaccustomed eccentric exercise. The large amount of HSP27, alphaB-crystallin, and HSP70 in the cytoskeletal/myofibrillar fraction after a repeated bout of exercise suggests a protective role as part of the repeated-bout effect.
Assuntos
Exercício Físico , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Contração Muscular , Músculo Esquelético/metabolismo , Sarcômeros/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Adulto , Biópsia , Western Blotting , Celecoxib , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Citosol/metabolismo , Desmina/metabolismo , Cotovelo , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Choque Térmico , Humanos , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Chaperonas Moleculares , Força Muscular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/ultraestrutura , Miofibrilas/metabolismo , Transporte Proteico , Pirazóis/administração & dosagem , Sarcômeros/efeitos dos fármacos , Sarcômeros/ultraestrutura , Sulfonamidas/administração & dosagem , Fatores de Tempo , Adulto JovemRESUMO
Attention-deficit/hyperactivity disorder (ADHD) is the most common neurobehavioural disorder among children. ADHD children are hyperactive, impulsive and have problems with sustained attention. These cardinal features are also present in the best validated animal model of ADHD, the spontaneously hypertensive rat (SHR), which is derived from the Wistar Kyoto rat (WKY). Current theories of ADHD relate symptom development to factors that alter learning. N-methyl-D-aspartate receptor (NMDAR) dependent long term changes in synaptic efficacy in the mammalian CNS are thought to represent underlying cellular mechanisms for some forms of learning. We therefore hypothesized that synaptic abnormality in excitatory, glutamatergic synaptic transmission might contribute to the altered behavior in SHRs. We studied physiological and anatomical aspects of hippocampal CA3-to-CA1 synapses in age-matched SHR and WKY (controls). Electrophysiological analysis of these synapses showed reduced synaptic transmission (reduced field excitatory postsynaptic potential for a defined fiber volley size) in SHR, whereas short-term forms of synaptic plasticity, like paired-pulse facilitation, frequency facilitation, and delayed response enhancement were comparable in the two genotypes, and long-term potentiation (LTP) of synaptic transmission was of similar magnitude. However, LTP in SHR was significantly reduced (by 50%) by the NR2B specific blocker CP-101,606 (10 microM), whereas the blocker had no effect on LTP magnitude in the control rats. This indicates that the SHR has a functional predominance of NR2B, a feature characteristic of early developmental stages in these synapses. Quantitative immunofluorescence and electron microscopic postembedding immunogold cytochemistry of the three major NMDAR subunits (NR1, NR2A; and NR2B) in stratum radiatum spine synapses revealed no differences between SHR and WKY. The results indicate that functional impairments in glutamatergic synaptic transmission may be one of the underlying mechanisms leading to the abnormal behavior in SHR, and possibly in human ADHD.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/genética , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores/fisiologia , Genótipo , Hipocampo/fisiopatologia , Hipocampo/ultraestrutura , Potenciação de Longa Duração/genética , Potenciação de Longa Duração/fisiologia , Masculino , Subunidades Proteicas/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/genética , Especificidade da Espécie , Sinapses/ultraestruturaRESUMO
There is now growing evidence that astrocytes, like neurons, can release transmitters. One transmitter that in a vast number of studies has been shown to be released from astrocytes is glutamate. Although asytrocytic glutamate may be released by several mechanisms, the evidence in favor of exocytosis is most compelling. Astrocytes may respond to neuronal activity by such exocytotic release of glutamate. The astrocyte derived glutamate can in turn activate neuronal glutamate receptors, in particular N-methyl-D-aspartate (NMDA) receptors. Here we review the morphological data supporting that astrocytes possess the machinery for exocytosis of glutamate. We describe the presence of small synaptic-like microvesicles, SNARE proteins and vesicular glutamate transporters in astrocytes, as well as NMDA receptors situated in vicinity of the astrocytic vesicles.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Exocitose/fisiologia , Ácido Glutâmico/metabolismo , Vesículas Secretórias/metabolismo , Animais , Astrócitos/ultraestrutura , Encéfalo/ultraestrutura , Comunicação Celular/fisiologia , Humanos , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas SNARE/metabolismo , Vesículas Secretórias/ultraestrutura , Proteínas Vesiculares de Transporte de Glutamato/metabolismoRESUMO
Adenosine and ATP, via their specific P1 and P2 receptors, modulate a wide variety of cellular and tissue functions, playing a neuroprotective or neurodegenerative role in brain damage conditions. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, recent data suggest the existence of a heterodimerization and a functional interaction between P1 and P2 receptors in the brain. In particular, interactions of adenosine A1 and P2Y1 receptors may play important roles in the purinergic signalling cascade. In the present work, we investigated the subcellular localization/co-localization of the receptors and their functional cross-talk at the membrane level in Wistar rat hippocampus. This is a particularly vulnerable brain area, which is sensitive to adenosine- and ATP-mediated control of glutamatergic transmission. The postembedding immunogold electron microscopy technique showed that the two receptors are co-localized at the synaptic membranes and surrounding astroglial membranes of glutamatergic synapses. To investigate the functional cross-talk between the two types of purinergic receptors, we evaluated the reciprocal effects of their activation on their G protein coupling. P2Y1 receptor stimulation impaired the potency of A1 receptor coupling to G protein, whereas the stimulation of A1 receptors increased the functional responsiveness of P2Y1 receptors. The results demonstrated an A1-P2Y1 receptor co-localization at glutamatergic synapses and surrounding astrocytes and a functional interaction between these receptors in hippocampus, suggesting ATP and adenosine can interact in purine-mediated signalling. This may be particularly important during pathological conditions, when large amounts of these mediators are released.
Assuntos
Hipocampo/fisiologia , Receptor Cross-Talk/fisiologia , Receptor A1 de Adenosina/fisiologia , Receptores Purinérgicos P2/fisiologia , Animais , Astrócitos/fisiologia , Western Blotting , Química Encefálica , Interpretação Estatística de Dados , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Trifosfato/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Membranas/química , Membranas/metabolismo , Microscopia Imunoeletrônica , Inclusão em Plástico , Ratos , Ratos Wistar , Receptores Purinérgicos P2Y1 , Transdução de Sinais/fisiologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
The vertebrate neuromuscular junction (NMJ) is known to be a cholinergic synapse at which acetylcholine (ACh) is released from the presynaptic terminal to act on postsynaptic nicotinic ACh receptors. There is now growing evidence that glutamate, which is the main excitatory transmitter in the CNS and at invertebrate NMJs, may have a signaling function together with ACh also at the vertebrate NMJ. In the CNS, the extracellular concentration of glutamate is kept at a subtoxic level by Na(+)-driven high-affinity glutamate transporters located in plasma membranes of astrocytes and neurons. The glutamate transporters are also pivotal for shaping glutamate receptor responses at synapses. In order to throw further light on the potential role of glutamate as a cotransmitter at the NMJ we used high-resolution immunocytochemical methods to investigate the localization of the plasma membrane glutamate transporters GLAST (glutamate aspartate transporter) and GLT (glutamate transporter 1) in rat and mice NMJ regions. Confocal laser-scanning immunocytochemistry showed that GLT is restricted to the NMJ in rat and mouse skeletal muscle. Lack of labeling signal in knock-out mice confirmed that the immunoreactivity observed at the NMJ was specific for GLT. GLAST was also localized at the NMJ in rat but not detected in mouse NMJ (while abundant in mouse brain). Post-embedding electron microscopic immunocytochemistry and quantitative analyses in rat showed that GLAST and GLT are enriched in the junctional folds of the postsynaptic membrane at the NMJ. GLT was relatively higher in the slow-twitch muscle soleus than in the fast-twitch muscle extensor digitorum longus, whereas GLAST was relatively higher in extensor digitorum longus than in soleus. The findings show--together with previous demonstration of vesicular glutamate, a vesicular glutamate transporter and glutamate receptors--that mammalian NMJs contain the machinery required for synaptic release and action of glutamate. This indicates a signaling role for glutamate at the normal NMJ and provides a basis for the ability of denervated muscle to be reinnervated by glutamatergic axons from the CNS.
Assuntos
Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Neurônios Motores/metabolismo , Junção Neuromuscular/metabolismo , Membranas Sinápticas/metabolismo , Animais , Transportador 1 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Imunoeletrônica , Neurônios Motores/ultraestrutura , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/inervação , Junção Neuromuscular/ultraestrutura , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Especificidade da Espécie , Membranas Sinápticas/ultraestrutura , Transmissão Sináptica/fisiologiaRESUMO
Intercellular monocarboxylate transport is important, particularly in tissues with high energy demands, such as brain and muscle. In skeletal muscle, it is well established that glycolytic fast twitch muscle fibers produce lactate, which is transported out of the cell through the monocarboxylate transporter (MCT) 4. Lactate is then taken up and oxidized by the oxidative slow twitch muscle fibers, which express MCT1. In the brain it is still questioned whether lactate produced in astrocytes is taken up and oxidized by neurons upon activation. Several studies have reported that astrocytes express MCT4, whereas neurons express MCT2. By comparing the localizations of MCTs in oxidative and glycolytic compartments I here give support to the idea that there is a lactate shuttle in the brain similar to that in muscle. This conclusion is based on studies in rodents using high resolution immunocytochemical methods at the light and electron microscopical levels.
Assuntos
Encéfalo/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Músculos/metabolismo , Neurônios/metabolismo , AnimaisRESUMO
The monocarboxylate transporters 1 and 4 are expressed in brain as well as in skeletal muscle and play important roles in the energy metabolism of both tissues. In brain, monocarboxylate transporter 1 occurs in astrocytes, ependymocytes, and endothelial cells while monocarboxylate transporter 4 appears to be restricted to astrocytes. In muscle, monocarboxylate transporter 1 is enriched in oxidative muscle fibers whereas monocarboxylate transporter 4 is expressed in all fibers, with the lowest levels in oxidative fiber types. The mechanisms regulating monocarboxylate transporter 1 and monocarboxylate transporter 4 expression are not known. We hypothesized that the expression of these transporters would be sensitive to long term changes in metabolic activity level. This hypothesis can be tested in rat skeletal muscle, where permanent changes in activity level can be induced by cross-reinnervation. We transplanted motor axons originally innervating the fast-twitch extensor digitorum longus muscle to the slow-twitch soleus muscle and vice versa. Four months later, microscopic analysis revealed transformation of muscle fiber types in the cross-reinnervated muscles. Western blot analysis showed that monocarboxylate transporter 1 was increased by 140% in extensor digitorum longus muscle and decreased by 30% in soleus muscle after cross-reinnervation. In contrast, cross-reinnervation induced a 62% decrease of monocarboxylate transporter 4 in extensor digitorum longus muscle and a 1300% increase in soleus muscle. Our findings show that cross-reinnervation causes pronounced changes in the expression levels of monocarboxylate transporter 1 and monocarboxylate transporter 4, probably as a direct consequence of the new pattern of nerve impulses. The data indicate that the mode of innervation dictates the expression of monocarboxylate transporter proteins in the target cells and that the change in monocarboxylate transporter isoform profile is an integral part of the muscle fiber transformation that occurs after cross-reinnervation. Our findings support the hypothesis that the expression of monocarboxylate transporter 1 and monocarboxylate transporter 4 in excitable tissues is regulated by activity.
Assuntos
Transportadores de Ácidos Monocarboxílicos/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Simportadores/metabolismo , Animais , Axônios/fisiologia , Axônios/transplante , Comunicação Celular/fisiologia , Denervação , Regulação para Baixo/fisiologia , Neurônios Motores/fisiologia , Neurônios Motores/transplante , Contração Muscular/fisiologia , Junção Neuromuscular/metabolismo , Nervos Periféricos/fisiologia , Nervos Periféricos/transplante , Ratos , Regulação para Cima/fisiologiaRESUMO
Previous findings, mainly in in vitro systems, have shown that the density of vesicles and the synaptic efficacy at excitatory synapses are reduced in the absence of synapsins, despite the fact that transgenic mice lacking synapsins develop an epileptic phenotype. Here we study glutamate receptors by quantitative immunoblotting and by quantitative electron microscopic postembedding immunocytochemistry in hippocampus of perfusion fixed control wild type and double knock-out mice lacking synapsins I and II. In wild type hippocampus the densities of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits were higher (indicated for glutamate receptor subunit 1, highly significant for glutamate receptor subunits 2/3) in mossy fiber-to-cornu ammonis 3 pyramidal cell synapses than in the Schaffer collateral/commissural-to-cornu ammonis 1 pyramidal cell synapses, the two synapse categories that carry the main excitatory throughput of the hippocampus. The opposite was true for N-methyl-D-aspartate receptors. The difference in localization of glutamate receptor subunit 1 receptor subunits was increased in the double knock-out mice while there was no change in the overall expression of the glutamate receptors in hippocampus as shown by quantitative Western blotting. The increased level of glutamate receptor subunit 1 at the mossy fiber-to-cornu ammonis 3 pyramidal cell synapse may result in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors with reduced proportions of glutamate receptor subunit 2, and hence increased Ca2+ influx, which could cause increased excitability despite of impaired synaptic function (cf. [Krestel HE, Shimshek DR, Jensen V, Nevian T, Kim J, Geng Y, Bast T, Depaulis A, Schonig K, Schwenk F, Bujard H, Hvalby O, Sprengel R, Seeburg PH (2004) A genetic switch for epilepsy in adult mice. J Neurosci 24:10568-10578]), possibly underlying the seizure proneness in the synapsin double knock-out mice. In addition, the tendency to increased predominance of N-methyl-d-aspartate receptors at the main type of excitatory synapse onto cornu ammonis 1 pyramidal cells might contribute to the seizure susceptibility of the synapsin deficient mice. The results showed no significant changes in the proportion of 'silent' Schaffer collateral/commissural synapses lacking alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors or in the synaptic membrane size, indicating that plasticity involving these parameters is not preferentially triggered due to lack of synapsins.
Assuntos
Hipocampo/patologia , Receptores de Glutamato/metabolismo , Receptores de Glutamato/ultraestrutura , Sinapses/ultraestrutura , Sinapsinas/deficiência , Análise de Variância , Animais , Western Blotting/métodos , Contagem de Células/métodos , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica/métodos , Receptores de Glutamato/classificação , Sinapses/classificaçãoRESUMO
Monocarboxylate transporters (MCTs) play an important role in the metabolism of all cells. They mediate the transport of lactate and pyruvate but also some other substrates such as ketone bodies. It has been proposed that glial cells release monocarboxylates to fuel neighbouring neurons. A key element in this hypothesis is the existence of neuronal MCTs. Amongst the three MCTs known to be expressed in the brain (MCT1, 2 and 4) only MCT2 has been found in neurons. Here we have studied the expression pattern of MCT2 during postnatal development. By use of immunoperoxidase and double immunofluorescence microscopy we report that neuronal MCT2 occurs in most brain areas, including the hippocampus and cerebellum, from birth to adult. MCT2 is also expressed in specific subpopulations of astrocytes. Neuronal MCT2 is most abundant in the first 3 postnatal weeks and thereafter decreases toward adulthood. In contrast to MCT2, MCT4 is exclusively present in astroglia during all stages of development. Furthermore, MCT4 expression is very low at birth and reaches adult level by P14. Our results are consistent with previous data suggesting that in the immature brain much of the energy demand is met by monocarboxylates and ketone bodies.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/anatomia & histologia , Encéfalo/crescimento & desenvolvimento , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/genética , Proteína Básica da Mielina/metabolismo , Gravidez , Ratos , Ratos WistarRESUMO
BACKGROUND: Cardiac metabolism becomes more dependent on carbohydrates in congestive heart failure (CHF), and lactate may be used as an important respiratory substrate. Monocarboxylate transporter 1 (MCT1) promotes cotransport of lactate and protons into and out of heart cells and conceivably flux of lactate between cells, because it is abundantly present in the intercalated disk. METHODS AND RESULTS: Six weeks after induction of myocardial infarction (MI) in Wistar rats, left ventricular end-diastolic pressures were >15 mm Hg, signifying CHF. MCT1 and connexin43 protein levels in CHF were 260% and 20%, respectively, of those in sham-operated animals (Sham), and the corresponding mRNA signals were 181% and not significantly changed, respectively. Confocal laserscan immunohistochemistry and quantitative immunogold cytochemistry showed that MCT1 density was much higher in CHF than in Sham both at the surface membrane and in the intercalated disk. In CHF, a novel intracellular pool of MCT1 appeared to be associated with cisternae, some close to the T tubules. In contrast, connexin43 particles, seen exclusively at gap junctions, were substantially fewer. Maximum lactate uptake was 107+/-15 mmol. L(-1). min(-1) in CHF and 42+/-6 mmol. L(-1). min(-1) in Sham cells (P<0.05). The K(m) values were between 7 and 9 mmol/L (P=NS). CONCLUSIONS: In cardiomyocytes from CHF rats, (1) the amount of functional MCT1 in the sarcolemma, including in the intercalated disk, is increased several-fold; (2) a new intracellular pool of MCT1 appears; (3) another disk protein, connexin43, is much reduced; and (4) increased reliance on lactate and other monocarboxylates (eg, pyruvate) could provide tight metabolic control of high-energy phosphates.
Assuntos
Proteínas de Transporte/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/química , Animais , Northern Blotting , Western Blotting , Proteínas de Transporte/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/fisiopatologia , Lactatos/farmacocinética , Microscopia Confocal , Microscopia Eletrônica , Transportadores de Ácidos Monocarboxílicos , Miocárdio/patologia , Miocárdio/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Regulação para CimaRESUMO
Confocal immunofluorescence microscopy showed strong monocarboxylate transporter 2 (MCT2) labeling of Purkinje cell bodies and punctate labeling in the molecular layer. By immunogold cytochemistry, it could be demonstrated that the MCT2 immunosignal was concentrated at postsynaptic densities of parallel fiber-Purkinje cell synapses. The distribution of MCT2 transporters within the individual postsynaptic densities mimicked that of the delta2 glutamate receptor, as shown by use of two different gold-particle sizes. The MCT2 distribution was also compared with the distributions of other monocarboxylate transporters (MCT1 and MCT4). The MCT1 immunolabeling was localized in the endothelial cells, while MCT4 immunogold particles were associated with glial profiles, including those abutting the synaptic cleft of the parallel fiber-spine synapses. The postsynaptic density (PSD) molecules identified so far can be divided into five classes: receptors, their anchoring molecules, molecules involved in signal transduction, ion channels, and attachment proteins. Here, we provide evidence that this list of molecules must now be extended to comprise an organic molecule transporter: the monocarboxylate transporter MCT2. The present data suggest that MCT2 has specific transport functions related to the synaptic cleft and that this transporter may allow an influx of lactate derived from perisynaptic glial processes. The expression of MCT2 in synaptic membranes may allow energy supply to be tuned to the excitatory drive.