Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing

When administered either systemically or centrally, glucose is a potent enhancer of memory processes. Measures of glucose levels in extracellular fluid in the rat hippocampus during memory tests reveal that these levels are dynamic, decreasing in response to memory tasks and loads; exogenous glucose blocks these decreases and enhances memory. The present experiments test the hypothesis that glucose enhancement of memory is mediated by glycogen storage and then metabolism to lactate in astrocytes, which provide lactate to neurons as an energy substrate. Sensitive bioprobes were used to measure brain glucose and lactate levels in 1-sec samples. Extracellular glucose decreased and lactate increased while rats performed a spatial working memory task. Intrahippocampal infusions of lactate enhanced memory in this task. In addition, pharmacological inhibition of astrocytic glycogenolysis impaired memory and this impairment was reversed by administration of lactate or glucose, both of which can provide lactate to neurons in the absence of glycogenolysis. Pharmacological block of the monocarboxylate transporter responsible for lactate uptake into neurons also impaired memory and this impairment was not reversed by either glucose or lactate. These findings support the view that astrocytes regulate memory formation by controlling the provision of lactate to support neuronal functions

Glucose Transporter 1 and Monocarboxylate Transporters 1, 2, and 4 Localization within the Glial Cells of Shark Blood-Brain-Barriers

Although previous studies showed that glucose is used to support the metabolic activity of the cartilaginous fish brain, the distribution and expression levels of glucose transporter (GLUT) isoforms remained undetermined. Optic/ultrastructural immunohistochemistry approaches were used to determine the expression of GLUT1 in the glial blood-brain barrier (gBBB). GLUT1 was observed solely in glial cells; it was primarily located in end-feet processes of the gBBB. Western blot analysis showed a protein with a molecular mass of 50 kDa, and partial sequencing confirmed GLUT1 identity. Similar approaches were used to demonstrate increased GLUT1 polarization to both apical and basolateral membranes in choroid plexus epithelial cells. To explore monocarboxylate transporter (MCT) involvement in shark brain metabolism, the expression of MCTs was analyzed. MCT1, 2 and 4 were expressed in endothelial cells; however, only MCT1 and MCT4 were present in glial cells. In neurons, MCT2 was localized at the cell membrane whereas MCT1 was detected within mitochondria. Previous studies demonstrated that hypoxia modified GLUT and MCT expression in mammalian brain cells, which was mediated by the transcription factor, hypoxia inducible factor-1. Similarly, we observed that hypoxia modified MCT1 cellular distribution and MCT4 expression in shark telencephalic area and brain stem, confirming the role of these transporters in hypoxia adaptation. Finally, using three-dimensional ultrastructural microscopy, the interaction between glial end-feet and leaky blood vessels of shark brain was assessed in the present study. These data suggested that the brains of shark may take up glucose from blood using a different mechanism than that used by mammalian brains, which may induce astrocyte-neuron lactate shuttling and metabolic coupling as observed in mammalian brain. Our data suggested that the structural conditions and expression patterns of GLUT1, MCT1, MCT2 and MCT4 in shark brain may establish the molecular foundation of metabolic coupling between glia and neurons

Overview of the Proton-coupled MCT (SLC16A) Family of Transporters: Characterization, Function and Role in the Transport of the Drug of Abuse γ-Hydroxybutyric Acid

The transport of monocarboxylates, such as lactate and pyruvate, is mediated by the SLC16A family of proton-linked membrane transport proteins known as monocarboxylate transporters (MCTs). Fourteen MCT-related genes have been identified in mammals and of these seven MCTs have been functionally characterized. Despite their sequence homology, only MCT1–4 have been demonstrated to be proton-dependent transporters of monocarboxylic acids. MCT6, MCT8 and MCT10 have been demonstrated to transport diuretics, thyroid hormones and aromatic amino acids, respectively. MCT1–4 vary in their regulation, tissue distribution and substrate/inhibitor specificity with MCT1 being the most extensively characterized isoform. Emerging evidence suggests that in addition to endogenous substrates, MCTs are involved in the transport of pharmaceutical agents, including γ-hydroxybuytrate (GHB), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins), salicylic acid, and bumetanide. MCTs are expressed in a wide range of tissues including the liver, intestine, kidney and brain, and as such they have the potential to impact a number of processes contributing to the disposition of xenobiotic substrates. GHB has been extensively studied as a pharmaceutical substrate of MCTs; the renal clearance of GHB is dose-dependent with saturation of MCT-mediated reabsorption at high doses. Concomitant administration of GHB and l-lactate to rats results in an approximately two-fold increase in GHB renal clearance suggesting that inhibition of MCT1-mediated reabsorption of GHB may be an effective strategy for increasing renal and total GHB elimination in overdose situations. Further studies are required to more clearly define the role of MCTs on drug disposition and the potential for MCT-mediated detoxification strategies in GHB overdose

Identification of novel common variants associated with chronic pain using conditional false discovery rate analysis with major depressive disorder and assessment of pleiotropic effects of LRFN5

Chronic pain is a complex trait that is moderately heritable and genetically, as well as phenotypically, correlated with major depressive disorder (MDD). Use of the conditional false discovery rate (cFDR) approach, which leverages pleiotropy identified from existing GWAS outputs, has been successful in discovering novel associated variants in related phenotypes. Here, genome-wide association study outputs for both von Korff chronic pain grade and for MDD were used to identify variants meeting a cFDR threshold for each outcome phenotype separately, as well as a conjunctional cFDR (ccFDR) threshold for both phenotypes together. Using a moderately conservative threshold, we identified a total of 11 novel single nucleotide polymorphisms (SNPs), six of which were associated with chronic pain grade and nine of which were associated with MDD. Four SNPs on chromosome 14 were associated with both chronic pain grade and MDD. SNPs associated only with chronic pain grade were located within SLC16A7 on chromosome 12. SNPs associated only with MDD were located either in a gene-dense region on chromosome 1 harbouring LINC01360, LRRIQ3, FPGT and FPGT-TNNI3K, or within/close to LRFN5 on chromosome 14. The SNPs associated with both outcomes were also located within LRFN5. Several of the SNPs on chromosomes 1 and 14 were identified as being associated with expression levels of nearby genes in the brain and central nervous system. Overall, using the cFDR approach, we identified several novel genetic loci associated with chronic pain and we describe likely pleiotropic effects of a recently identified MDD locus on chronic pain

Distribution of neuronal and metabolic markers in the human auditory cortex

SUMMARY The human auditory cortex, located on the supratemporal plane of the temporal lobe, is divided in a primary auditory area and several non-primary areas surrounding it. These different areas show anatomical and functional differences. Many studies have focussed on auditory areas in non-human primates, using investigation techniques such as electrophysiological recordings, tracing of neural connections, or immunohistochemical and histochemical staining. Some of these studies have suggested parallel and hierarchical organization of the cortical auditory areas as well as subcortical auditory relays. In humans, only few studies have investigated these regions immunohistochemically, but activation and lesion studies speak in favour of parallel and hierarchical organization, very similar to that of non-human primates. Calcium-binding proteins and metabolic markers were used to investigate possible correlates of hierarchical and parallel organization in man. Calcium-binding proteins, parvalbumin, calretinin and calbindin, modulate the concentration of intracellular free calcium ions and were found in distinct subpopulations of GABAergic neurons in non-human primates species. In our study, their distribution showed several differences between auditory areas: the primary auditory area was darkly stained for both parvalbumin and calbindin, and their expression rapidly decreased while moving away from the primary area. This staining pattern suggests a hierarchical organization of the areas, in which the more darkly stained areas could correspond to an earlier integration level and the areas showing light staining may correspond to higher level integration areas. Parallel organization of primary and non-primary auditory areas was suggested by the complementarity, within a given area, between parvalbumin and calbindin expression across layers. To investigate the possible differences in the energetic metabolism of the cortical auditory areas, several metabolic markers were used: cytochrome oxidase and LDH1 were used as oxidative metabolism markers and LDH5 was used as glycolytic metabolism marker. The results obtained show a difference in the expression of enzymes involved in oxidative metabolism between areas. In the primary auditory area the oxidative metabolism markers were maximally expressed in layer IV. In contrast, higher order areas showed maximal staining in supragranular layers. The expression of LDH5 varied in patches, but did not differ between the different hierarchical auditory areas. The distribution of the two LDH enzymes isoforms also provides information about cellular aspects of metabolic organization, since neurons expressed the LDH1 isoform whereas astrocytes express primarily LDH5, but some astrocytes also contained the LDH1 isoform. This cellular distribution pattern supports the hypothesis of the existence of an astrocyte-neuron lactate shuttle, previously suggested in rodent studies, and in particular of lactate transfer from astrocytes, which produce lactate from the glucose obtained from the circulation, to neurons that use lactate as energy substrate. In conclusion, the hypothesis of parallel and hierarchical organization of the auditory areas can be supported by CaBPs, cytochrome oxidase and LDH1 distribution. Moreover, the two LDHs cellular distribution pattern support the hypothesis of an astrocyte-neuron lactate shuttle in human cortex

Patterns of calcium-binding proteins support parallel and hierarchical organization of human auditory areas.

The human primary auditory cortex (AI) is surrounded by several other auditory areas, which can be identified by cyto-, myelo- and chemoarchitectonic criteria. We report here on the pattern of calcium-binding protein immunoreactivity within these areas. The supratemporal regions of four normal human brains (eight hemispheres) were processed histologically, and serial sections were stained for parvalbumin, calretinin or calbindin. Each calcium-binding protein yielded a specific pattern of labelling, which differed between auditory areas. In AI, defined as area TC [see C. von Economo and L. Horn (1930) Z. Ges. Neurol. Psychiatr.,130, 678-757], parvalbumin labelling was dark in layer IV; several parvalbumin-positive multipolar neurons were distributed in layers III and IV. Calbindin yielded dark labelling in layers I-III and V; it revealed numerous multipolar and pyramidal neurons in layers II and III. Calretinin labelling was lighter than that of parvalbumin or calbindin in AI; calretinin-positive bipolar and bitufted neurons were present in supragranular layers. In non-primary auditory areas, the intensity of labelling tended to become progressively lighter while moving away from AI, with qualitative differences between the cytoarchitectonically defined areas. In analogy to non-human primates, our results suggest differences in intrinsic organization between auditory areas that are compatible with parallel and hierarchical processing of auditory information

Patterns of calcium-binding proteins in human inferior colliculus: identification of subdivisions and evidence for putative parallel systems.

The subdivisions of human inferior colliculus are currently based on Golgi and Nissl-stained preparations. We have investigated the distribution of calcium-binding protein immunoreactivity in the human inferior colliculus and found complementary or mutually exclusive localisations of parvalbumin versus calbindin D-28k and calretinin staining. The central nucleus of the inferior colliculus but not the surrounding regions contained parvalbumin-positive neuronal somata and fibres. Calbindin-positive neurons and fibres were concentrated in the dorsal aspect of the central nucleus and in structures surrounding it: the dorsal cortex, the lateral lemniscus, the ventrolateral nucleus, and the intercollicular region. In the dorsal cortex, labelling of calbindin and calretinin revealed four distinct layers.Thus, calcium-binding protein reactivity reveals in the human inferior colliculus distinct neuronal populations that are anatomically segregated. The different calcium-binding protein-defined subdivisions may belong to parallel auditory pathways that were previously demonstrated in non-human primates, and they may constitute a first indication of parallel processing in human subcortical auditory structures

Distribution of the monocarboxylate transporter MCT2 in human cerebral cortex: an immunohistochemical study

The monocarboxylate transporter MCT2 belongs to a large family of membrane proteins involved in the transport of lactate, pyruvate and ketone bodies. Although its expression in rodent brain has been well documented, the presence of MCT2 in the human brain has been questioned on the basis of low mRNA abundance. In this study, the distribution of the monocarboxylate transporter MCT2 has been investigated in the cortex of normal adult human brain using an immunohistochemical approach. Widespread neuropil staining in all cortical layers was observed by light microscopy. Such a distribution was very similar in three different cortical areas investigated. At the cellular level, the expression of MCT2 could be observed in a large number of neurons, in fibers both in grey and white matter, as well as in some astrocytes, mostly localized in layer I and in the white matter. Double staining experiments combined with confocal microscopy confirmed the neuronal expression but also suggested a preferential postsynaptic localization of synaptic MCT2 expression. A few astrocytes in the grey matter appeared to exhibit MCT2 labelling but at low levels. Electron microscopy revealed strong MCT2 expression at asymmetric synapses in the postsynaptic density and also within the spine head but not in the presynaptic terminal. These data not only demonstrate neuronal MCT2 expression in human, but since a portion of it exhibits a distinct synaptic localization, it further supports a putative role for MCT2 in adjustment of energy supply to levels of activity

Expression of the monocarboxylate transporter MCT1 in the adult human brain cortex.

Distribution of the monocarboxylate transporter MCT1 has been investigated in the cortex of normal adult human brain. Similarly to the glucose transporter GLUT1 55 kDa isoform, MCT1 was found to be strongly expressed on blood vessels in all cortical layers. In addition, laminar analysis revealed intense MCT1 expression in the neuropil of layer IV in primary auditory (AI) and visual (VI) areas, while this expression was more homogeneous in the non-primary auditory area STA. The cellular distribution shows that MCT1 is strongly expressed by glial cells often associated with blood vessels that were identified as astrocytes. The observed distribution of MCT1 supports the concept that, under certain circumstances, monocarboxylates could be provided as energy substrates to the adult human brain. Moreover, the distinct laminar pattern of MCT1 expression between primary and non-primary cortical areas may reflect different types of neuronal activity requiring adequate supply of specific energy substrates

Histological validation of DW-MRI tractography in human postmortem tissue.

Despite several previous attempts, histological validation of diffusion-weighted magnetic resonance imaging (DW-MRI)-based tractography as true axonal fiber pathways remains difficult. In the present study, we establish a method to compare histological and tractography data precisely enough for statements on the level of single tractography pathways. To this end, we used carbocyanine dyes to trace connections in human postmortem tissue and aligned them to high-resolution DW-MRI of the same tissue processed within the diffusion tensor imaging (DTI) formalism. We provide robust definitions of sensitivity (true positives) and specificity (true negatives) for DTI tractography and characterize tractography paths in terms of receiver operating characteristics. With sensitivity and specificity rates of approximately 80%, we could show a clear correspondence between histological and inferred tracts. Furthermore, we investigated the effect of fractional anisotropy (FA) thresholds for the tractography and identified FA values between 0.02 and 0.08 as optimal in our study. Last, we validated the course of entire tractography curves to move beyond correctness determination based on pairs of single points on a tract. Thus, histological techniques, in conjunction with alignment and processing tools, may serve as an important validation method of DW-MRI on the level of inferred tractography projections between brain areas