Changes in ubiquitin immunoreactivity in developing rat brain: a putative role for ubiquitin and ubiquitin conjugates in dendrite outgrowth and differentiation.

The role of ubiquitin in development of the mammalian brain has been studied using a monoclonal antibody, RHUb1, specific for ubiquitin. Immunodevelopment of western blots of homogenate samples of the cerebral cortex, hippocampus and cerebellum prepared from animals of known postnatal age show marked developmental changes in conjugate level. Striking decreases in the level of a prominent conjugate of molecular weight 22,000, which is identified as ubiquitinated histone, are observed during the first postnatal week in the cerebral cortex and hippocampus, but not the cerebellum. A marked overall developmental decrease in the level of high-molecular-weight (> 40,000) ubiquitin conjugates which occurs predominantly during the third, but also the fourth, postnatal week is observed in all three regions. Immunocytochemical data obtained with the RHUb1 antibody show intense staining of neuronal perikarya, nuclei and dendrites in early postnatal cerebral cortex and hippocampus. Staining of pyramidal cell perikarya and dendrites is particularly prominent. The intensity of dendritic staining, particularly for the cerebral cortex, shows a striking decrease after postnatal day 14 and only faint dendritic staining is observed in the adult. In early postnatal cerebellum, immunoreactivity is predominantly nuclear, though some staining of the proximal regions of Purkinje cell dendrites is observed between postnatal days 4 and 19. As with the cerebral cortex and hippocampus, most of the ubiquitin reactivity is lost in adult animals. The loss of dendritic staining, particularly in the cerebral cortex, correlates with the decrease in the level of high-molecular-weight ubiquitin conjugates observed on the western blots. Immunodevelopment of western blots of a range of subcellular fractions prepared from developing rat forebrain shows that the developmental decrease in the level of high-molecular-weight ubiquitin conjugates is not uniform for all fractions. The decrease in conjugate level is most marked for the cell-soluble, mitochondrial and detergent-insoluble cytoskeletal fractions. Taken overall, the data suggest a role for ubiquitin in dendrite outgrowth and arborization, loss of dendritic ubiquitin immunoreactivity correlating with completion of these processes.

VGLUT1 is Present in Cortical, Hippocampal and Striatal Astrocytes

The last decade has presented studies providing evidence for astrocytic exocytosis of glutamate potentiating nerve signals. To make further investigations into this astrocytic attribute we investigated the localization of the vesicular glutamate transporter 1 (VGLUT1) in small processes of astrocytes close to glutamatergic terminals in frontal cortex, striatum, molecular layer of hippocampus and stratum radiatum of hippocampus. According to the importance of VGLUT1 in glutamate exocytosis the presence of VGLUT1 in astrocytic processes indicates the ability to exocytose glutamate. METHODS: For qualitative analysis we used immunoflourescence histochemistry. Sections from rat frontal cortex, striatum, molecular layer of hippocampus and stratum radiatum of hippocampus were labeled with antibodies against glutamine synthetase (an astrocytic marker) and VGLUT1. Z-stacks of 4.5-5 lm obtained by confocal microscopy from each section were deconvolved and 3D reconstructed in Amira. Small astrocytic processes were analysed for the presence of VGLUT1 inside the processes. The quantitative analysis was done by immunogold labeling. Ultrathin sections from each brain region were labeled for GLT (an astrocytic marker) and VGLUT1. Pictures obtained by electron microscopy were analysed and the point density (gold particles/nm2) for VGLUT1 in astrocytic processes was measured. RESULTS: Using confocal 3D reconstructions we were qualitatively able to identify VGLUT1 within small processes of astrocytes in all four brain regions. Reflecting our qualitative findings the electron microscopical immunogold quantifications showed a significant density of gold particles signaling VGLUT1 in astrocytic processes in all four brain regions. CONCLUSION: We extend the results of previous studies on glutamate release from astrocytes, which have focused on the hippocampus, proposing that astrocytic exocytosis of glutamate is a global phenomenon in the brain.

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain.

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.

Formation of a spatial memory trace : a behavioral, cellular and molecular study

THESIS ABSTRACTThis thesis project was aimed at studying the molecular mechanisms underlying learning and memory formation, in particular as they relate to the metabolic coupling between astrocytes and neurons. For that, changes in the metabolic activity of different mice brain regions after 1 or 9 days of training in an eight-arm radial maze were assessed by (14C) 2-deoxyglucose (2DG) autoradiography. Significant differences in the areas engaged during the behavioral task at day 1 (when animals are confronted for the first time to the learning task) and at day 9 (when animals are highly performing) have been identified. These areas include the hippocampus, the fornix, the parietal cortex, the laterodorsal thalamic nucleus and the mammillary bodies at day 1 ; and the anterior cingulate, the retrosplenial cortex and the dorsal striatum at day 9. Two of these cerebral regions (those presenting the greatest changes at day 1 and day 9: the hippocampus and the retrosplenial cortex, respectively) were microdissected by laser capture microscopy and selected genes related to neuron-glia metabolic coupling, glucose metabolism and synaptic plasticity were analyzed by RT-PCR. 2DG and gene expression analysis were performed at three different times: 1) immediately after the end of the behavioral paradigm, 2) 45 minutes and 3) 6 hours after training. The main goal of this study was the identification of the metabolic adaptations following the learning task. Gene expression results demonstrate that the learning task profoundly modulates the pattern of gene expression in time, meaning that these two cerebral regions with high 2DG signal (hippocampus and retrosplenial cortex) have adapted their metabolic molecular machinery in consequence. Almost all studied genes show a higher expression in the hippocampus at day 1 compared to day 9, while an increased expression was found in the retrosplenial cortex at day 9. We can observe these molecular adaptations with a short delay of 45 minutes after the end of the task. However, 6 hours after training a high gene expression was found at day 9 (compared to day 1) in both regions, suggesting that only one day of training is not sufficient to detect transcriptional modifications several hours after the task. Thus, gene expression data match 2DG results indicating a transfer of information in time (from day 1 to day 9) and in space (from the hippocampus to the retrosplenial cortex), and this at a cellular and a molecular level. Moreover, learning seems to modify the neuron-glia metabolic coupling, since several genes involved in this coupling are induced. These results also suggest a role of glia in neuronal plasticity.RESUME DU TRAVAIL DE THESECe projet de thèse a eu pour but l'étude des mécanismes moléculaires qui sont impliqués dans l'apprentissage et la mémoire et, en particulier, à les mettre en rapport avec le couplage métabolique existant entre les astrocytes et les neurones. Pour cela, des changements de l'activité métabolique dans différentes régions du cerveau des souris après 1 ou 9 jours d'entraînement dans un labyrinthe radial à huit-bras ont été évalués par autoradiographie au 2-désoxyglucose (2DG). Des différences significatives dans les régions engagées pendant la tâche comportementale au jour 1 (quand les animaux sont confrontés pour la première fois à la tâche) et au jour 9 (quand les animaux ont déjà appris) ont été identifiés. Ces régions incluent, au jour 1, l'hippocampe, le fornix, le cortex pariétal, le noyau thalamic laterodorsal et les corps mamillaires; et, au jour 9, le cingulaire antérieur, le cortex retrosplenial et le striatum dorsal. Deux de ces régions cérébrales (celles présentant les plus grands changements à jour 1 et à jour 9: l'hippocampe et le cortex retrosplenial, respectivement) ont été découpées par microdissection au laser et quelques gènes liés au couplage métabolique neurone-glie, au métabolisme du glucose et à la plasticité synaptique ont été analysées par RT-PCR. L'étude 2DG et l'analyse de l'expression de gènes ont été exécutés à trois temps différents: 1) juste après entraînement, 2) 45 minutes et 3) 6 heures après la fin de la tâche. L'objectif principal de cette étude était l'identification des adaptations métaboliques suivant la tâche d'apprentissage. Les résultats de l'expression de gènes démontrent que la tâche d'apprentissage module profondément le profile d'expression des gènes dans le temps, signifiant que ces deux régions cérébrales avec un signal 2DG élevé (l'hippocampe et le cortex retrosplenial) ont adapté leurs « machines moléculaires » en conséquence. Presque tous les gènes étudiés montrent une expression plus élevée dans l'hippocampe au jour 1 comparé au jour 9, alors qu'une expression accrue a été trouvée dans le cortex retrosplenial au jour 9. Nous pouvons observer ces adaptations moléculaires avec un retard court de 45 minutes après la fin de la tâche. Cependant, 6 heures après l'entraînement, une expression de gènes élevée a été trouvée au jour 9 (comparé à jour 1) dans les deux régions, suggérant que seulement un jour d'entraînement ne suffit pas pour détecter des modifications transcriptionelles plusieurs heures après la tâche. Ainsi, les données d'expression de gènes corroborent les résultats 2DG indiquant un transfert d'information dans le temps (de jour 1 à jour 9) et dans l'espace (de l'hippocampe au cortex retrosplenial), et ceci à un niveau cellulaire et moléculaire. D'ailleurs, la tâche d'apprentissage semble modifier le couplage métabolique neurone-glie, puisque de nombreux gènes impliqués dans ce couplage sont induits. Ces observations suggèrent un rôle important de la glie dans les mécanismes de plasticité du système nerveux.

Stereologic estimates of total spinophilin-immunoreactive spine number in area 9 and the CA1 field: Relationship with the progression of Alzheimer's disease

The loss of presynaptic markers is thought to represent a strong pathologic correlate of cognitive decline in Alzheimer's disease (AD). Spinophilin is a postsynaptic marker mainly located to the heads of dendritic spines. We assessed total numbers of spinophilin-immunoreactive puncta. in the CA I and CA3 fields of hippocampus and area 9 in 18 elderly individuals with various degrees of cognitive decline. The decrease in spinophilin-immunoreactivity was significantly related to both Braak neurofibrillary tangle (NFT) staging and clinical severity but not A beta deposition staging. The total number of spinophilin-immunoreactive puncta in CA I field and area 9 were significantly related to MMSE scores and predicted 23.5 and 61.9% of its variability. The relationship between total number of spinophilin-immunoreactive puncta in CA I field and MMSE scores did not persist when adjusting for Braak NFT staging. In contrast, the total number of spinophilin-immunoreactive puncta in area 9 was still significantly related to the cognitive outcome explaining an extra 9.6% of MMSE and 25.6% of the Clinical Dementia Rating scores variability. Our data suggest that neocortical dendritic spine loss is an independent parameter to consider in AD clinicopathologic correlations.

Glucose-dependent insulinotropic polypeptide receptor knockout mice are impaired in learning, synaptic plasticity, and neurogenesis.

Glucose-dependent insulinotropic polypeptide (GIP) is a key incretin hormone, released from intestine after a meal, producing a glucose-dependent insulin secretion. The GIP receptor (GIPR) is expressed on pyramidal neurons in the cortex and hippocampus, and GIP is synthesized in a subset of neurons in the brain. However, the role of the GIPR in neuronal signaling is not clear. In this study, we used a mouse strain with GIPR gene deletion (GIPR KO) to elucidate the role of the GIPR in neuronal communication and brain function. Compared with C57BL/6 control mice, GIPR KO mice displayed higher locomotor activity in an open-field task. Impairment of recognition and spatial learning and memory of GIPR KO mice were found in the object recognition task and a spatial water maze task, respectively. In an object location task, no impairment was found. GIPR KO mice also showed impaired synaptic plasticity in paired-pulse facilitation and a block of long-term potentiation in area CA1 of the hippocampus. Moreover, a large decrease in the number of neuronal progenitor cells was found in the dentate gyrus of transgenic mice, although the numbers of young neurons was not changed. Together the results suggest that GIP receptors play an important role in cognition, neurotransmission, and cell proliferation.

Brain structure in asymptomatic FMR1 premutation carriers at risk for fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset movement disorder affecting FMR1 premutation carriers, is associated with cerebral and cerebellar lesions. The aim of this study was to test whether computational anatomy can detect similar patterns in asymptomatic FMR1 premutation carriers (mean age 46.7 years) with qualitatively normal -appearing grey and white matter on brain MRI. We used a multimodal imaging protocol to characterize brain anatomy by automated assessment of gray matter volume and white matter properties. Structural changes in the hippocampus and in the cerebellar motor network with decreased gray matter volume in lobule VI and white matter alterations of the corresponding afferent projections through the middle cerebellar peduncles are demonstrated. Diffuse subcortical white matter changes in both hemispheres, without corresponding gray matter alterations, are only identified through age × group interactions. We interpret the hippocampal fimbria and cerebellar changes as early alterations with a possible neurodevelopmental origin. In contrast, progression of the diffuse cerebral hemispheric white matter changes suggests a neurodegenerative process, leading to late-onset lesions, which may mark the imminent onset of FXTAS.

Distinct subcellular localization of beta-tubulin epitopes in the adult mouse brain.

A panel of monoclonal antibodies specific of alpha-tubulin (TU-01, TU-09) and beta-tubulin (TU-06, TU-13) subunits was used to study the location of N-terminal structural domains of tubulin in adult mouse brain. The specificity of antibodies was confirmed b immunoblotting experiments. Immunohistochemical staining of vibratome sections from cerebral cortex, cerebellum, hippocampus, and corpus callosum showed that antibodies TU-01, TU-09, and TU-13 reacted with neuronal and glial cells and their processes, whereas the TU-06 antibody stained only the perikarya. Dendrites and axons were either unstained or their staining was very weak. As the TU-06 epitope is located on the N-terminal structural domain of beta-tubulin, the observed staining pattern cannot be interpreted as evidence of a distinct subcellular localization of beta-tubulin isotypes or known post-translational modifications. The limited distribution of the epitope could, rather, reflect differences between the conformations of tubulin molecules in microtubules of somata and neurites or, alternatively, a specific masking of the corresponding region on the N-terminal domain of beta-tubulin by interacting protein(s) in dendrites and axons.

Cellular pathology of hilar neurons in Ammon's horn sclerosis.

In addition to functionally affected neuronal signaling pathways, altered axonal, dendritic, and synaptic morphology may contribute to hippocampal hyperexcitability in chronic mesial temporal lobe epilepsies (MTLE). The sclerotic hippocampus in Ammon's horn sclerosis (AHS)-associated MTLE, which shows segmental neuronal cell loss, axonal reorganization, and astrogliosis, would appear particularly susceptible to such changes. To characterize the cellular hippocampal pathology in MTLE, we have analyzed hilar neurons in surgical hippocampus specimens from patients with MTLE. Anatomically well-preserved hippocampal specimens from patients with AHS (n = 44) and from patients with focal temporal lesions (non-AHS; n = 20) were studied using confocal laser scanning microscopy (CFLSM) and electron microscopy (EM). Hippocampal samples from three tumor patients without chronic epilepsies and autopsy samples were used as controls. Using intracellular Lucifer Yellow injection and CFLSM, spiny pyramidal, multipolar, and mossy cells as well as non-spiny multipolar neurons have been identified as major hilar cell types in controls and lesion-associated MTLE specimens. In contrast, none of the hilar neurons from AHS specimens displayed a morphology reminiscent of mossy cells. In AHS, a major portion of the pyramidal and multipolar neurons showed extensive dendritic ramification and periodic nodular swellings of dendritic shafts. EM analysis confirmed the altered cellular morphology, with an accumulation of cytoskeletal filaments and increased numbers of mitochondria as the most prominent findings. To characterize cytoskeletal alterations in hilar neurons further, immunohistochemical reactions for neurofilament proteins (NFP), microtubule-associated proteins, and tau were performed. This analysis specifically identified large and atypical hilar neurons with an accumulation of low weight NFP. Our data demonstrate striking structural alterations in hilar neurons of patients with AHS compared with controls and non-sclerotic MTLE specimens. Such changes may develop during cellular reorganization in the epileptogenic hippocampus and are likely to contribute to the pathogenesis or maintenance of temporal lobe epilepsy.

Primary motor cortex involvement in Alzheimer disease.

In Alzheimer disease (AD) the involvement of entorhinal cortex, hippocampus, and associative cortical areas is well established. Regarding the involvement of the primary motor cortex the reported data are contradictory. In order to determine whether the primary motor cortex is involved in AD, the brains of 29 autopsy cases were studied, including, 17 cases with severe cortical AD-type changes with definite diagnoses of AD, 7 age-matched cases with discrete to moderate cortical AD-type changes, and 5 control cases without any AD-type cortical changes. Morphometric analysis of the cortical surface occupied by senile plaques (SPs) on beta-amyloid-immunostained sections and quantitative analysis of neurofibrillary tangles (NFTs) on Gallyas-stained sections was performed in 5 different cortical areas including the primary motor cortex. The percentage of cortical surface occupied by SPs was similar in all cortical areas, without significant difference and corresponded to 16.7% in entorhinal cortex, 21.3% in frontal associative, 16% in parietal associative, and 15.8% in primary motor cortex. The number of NFTs in the entorhinal cortex was significantly higher (41 per 0.4 mm2), compared with those in other cortical areas (20.5 in frontal, 17.9 in parietal and 11.5 in the primary motor cortex). Our findings indicate that the primary motor cortex is significantly involved in AD and suggest the appearance of motor dysfunction in late and terminal stages of the disease.

Intracisternal delivery of NFκB-inducible scAAV2/9 reveals locoregional neuroinflammation induced by systemic kainic acid treatment.

We have previously demonstrated disease-dependent gene delivery in the brain using an AAV vector responding to NFκB activation as a probe for inflammatory responses. This vector, injected focally in the parenchyma prior to a systemic kainic acid (KA) injection mediated inducible transgene expression in the hippocampus but not in the cerebellum, regions, respectively, known to be affected or not by the pathology. However, such a focal approach relies on previous knowledge of the model parameters and does not allow to predict the whole brain response to the disease. Global brain gene delivery would allow to predict the regional distribution of the pathology as well as to deliver therapeutic factors in all affected brain regions. We show that self-complementary AAV2/9 (scAAV2/9) delivery in the adult rat cisterna magna allows a widespread but not homogenous transduction of the brain. Indeed, superficial regions, i.e., cortex, hippocampus, and cerebellum were more efficiently transduced than deeper regions, such as striatum, and substantia nigra. These data suggest that viral particles penetration from the cerebrospinal fluid (CSF) into the brain is a limiting factor. Interestingly, AAV2/9-2YF a rationally designed capsid mutant (affecting surface tyrosines) increased gene transfer efficiency approximately fivefold. Neurons, astrocytes, and oligodendrocytes, but not microglia, were transduced in varying proportions depending on the brain region and the type of capsid. Finally, after a single intracisternal injection of scAAV2/9-2YF using the NFκB-inducible promoter, KA treatment induced transgene expression in the hippocampus and cortex but not in the cerebellum, corresponding to the expression of the CD11b marker of microglial activation. These data support the use of disease-inducible vectors administered in the cisterna magna as a tool to characterize the brain pathology in systemic drug-induced or transgenic disease models. However, further improvements are required to enhance viral particles penetration into the brain.

Genetic manipulation of adult-born hippocampal neurons rescues memory in a mouse model of Alzheimer's disease.

In adult mammals, neural progenitors located in the dentate gyrus retain their ability to generate neurons and glia throughout lifetime. In rodents, increased production of new granule neurons is associated with improved memory capacities, while decreased hippocampal neurogenesis results in impaired memory performance in several memory tasks. In mouse models of Alzheimer's disease, neurogenesis is impaired and the granule neurons that are generated fail to integrate existing networks. Thus, enhancing neurogenesis should improve functional plasticity in the hippocampus and restore cognitive deficits in these mice. Here, we performed a screen of transcription factors that could potentially enhance adult hippocampal neurogenesis. We identified Neurod1 as a robust neuronal determinant with the capability to direct hippocampal progenitors towards an exclusive granule neuron fate. Importantly, Neurod1 also accelerated neuronal maturation and functional integration of new neurons during the period of their maturation when they contribute to memory processes. When tested in an APPxPS1 mouse model of Alzheimer's disease, directed expression of Neurod1 in cycling hippocampal progenitors conspicuously reduced dendritic spine density deficits on new hippocampal neurons, to the same level as that observed in healthy age-matched control animals. Remarkably, this population of highly connected new neurons was sufficient to restore spatial memory in these diseased mice. Collectively our findings demonstrate that endogenous neural stem cells of the diseased brain can be manipulated to become new neurons that could allow cognitive improvement.

Irritability in pre-clinical Huntington's disease.

Irritability, together with depression and anxiety, form three salient clinical features of pre-symptomatic Huntington's disease (HD). To date, the understanding of irritability in HD suffers from a paucity of experimental data and is largely based on questionnaires or clinical anecdotes. Factor analysis suggests that irritability is related to impulsivity and aggression and is likely to engage the same neuronal circuits as these behaviours, including areas such as medial orbitofrontal cortex (OFC) and amygdala. 16 pre-symptomatic gene carriers (PSCs) and 15 of their companions were asked to indicate the larger of two squares consecutively shown on a screen while undergoing functional magnetic resonance imaging (fMRI). Despite correct identification of the larger square, participants were often told that they or their partner had given the wrong answer. Size differences were subtle to make negative feedback credible but detectable. Although task performance, baseline irritability, and reported task-induced irritation were the same for both groups, fMRI revealed distinct neuronal processing in those who will later develop HD. In controls but not PSCs, task-induced irritation correlated positively with amygdala activation and negatively with OFC activation. Repetitive negative feedback induced greater amygdala activations in controls than PSCs. In addition, the inverse functional coupling between amygdala and OFC was significantly weaker in PSCs compared to controls. Our results argue that normal emotion processing circuits are disrupted in PSCs via attenuated modulation of emotional status by external or internal indicators. At later stages, this dysfunction may increase the risk for developing recognised, HD-associated, psychiatric symptoms such as irritability.

Contribution à l'étude neuroanatomique de systèmes enzymatiques impliqués dans le métabolisme énergétique cérébral

RESUME Il a longtemps été admis que le glucose était le principal, sinon le seul substrat du métabolisme énergétique cérébral. Néanmoins, des études récentes indiquent que dans des situations particulières, d'autres substrats peuvent être employés. C'est le cas des monocarboxylates (lactate et pyruvate principalement). Bien que la barrière hématoencéphalique soit peu perméable à ces molécules, elles deviennent néanmoins des substrats possibles si elles sont produites localement. Les deux systèmes enzymatiques pivots des voies glycolytiques et oxydatives sont la lactate déshydrogénase (LDH, EC 1.1.1.27) qui catalyse l'interconversion du pyruvate et du lactate et le complexe pyruvate déshydrogénase qui catalyse la conversion irréversible du pyruvate en acétyl-CoA qui entre dans la respiration mitochondriale. Nous avons étudié la localisation, tant régionale que cellulaire, des isoformes LDH-1, LDH-5 et PDHEla dans le cerveau du chat et dé l'homme au moyen de diverses techniques histologiques. Dans un premier temps, des investigations par hybridation in situ au moyen d'oligosondes marquées au 33P sur de coupes de cerveau de chat ont permis de montrer une différence de l'expression des enzymes à vocation oxydative (LDH-1 et PDHA1, le gène codant pour la protéine PDHEIa) par rapport à LDH-5, isoforme qui catalyse préférentiellement la formation de lactate. LDH-1 et PDHA 1 ont des distributions similaires et sont enrichies dans de nombreuses structures cérébrales, comme l'hippocampe, de nombreux noyaux thalamiques et des structures pontiques. Le cortex cérébral exhibe également une expression importante de LDH-1 et PDH. LDH-5 a par contre une expression largement plus diffuse à travers le cerveau, bien que l'on trouve néanmoins un enrichissement plus important dans l'hippocampe. Ces résultats sont en accord avec les observations que nous avons précédemment publiées chez le rongeur pour LDH-1 et LDH-5 (Laughton et collaborateurs, 2000). Des analyses par PCR en temps réel ont confirmé que dans certaines régions, LDH-1 est exprimée de façon nettement plus importante que LDH-5. Dans un deuxième temps, nous avons appliqué sur des coupes histologiques d'hippocampe et de cortex occipital humain post-mortem des anticorps monoclonaux spécifiques de l'isoforme LDH-5 et la sous-unité PDHela du complexe pyruvate déshydrogénase. Là aussi, les immunoréactions révèlent une ségrégation régionale mais aussi cellulaire des deux enzymes. Dans les deux régions étudiées, LDH-5 est localisée exclusivement dans les astrocytes. Dans le cortex occipital, la matière blanche et également la couche I corticale sont immunopositives pour LDH-5. Dans l'hippocampe, le CA4 et l'alveus exhibe l'immunomarquage le plus intense pour LDH-5. Seuls des neurones (à de rares exceptions quelques astrocytes) sont immunopositifs à l'anticorps monoclonal dirigé contre PDHela. La couche IV du cortex occipital présente la plus forte immunoréaction. Dans l'hippocampe, une immunoréactivité est observée dans le stratum granulosum et à travers la région CA1 jusqu'à la région CA3. L'ensemble de ces résultats montre une hétérogénéité métabolique dans le cerveau et étaye l'hypothèse "astrocyte-neurone lactate shuttle" (ANL5) (Bittar et collaborateurs, 1996; Magistretti et Pellerin, 1999) qui propose que les astrocytes fournissent aux neurones activés du lactate comme substrat alternatif de leur métabolisme énergétique. ABSTRACT For a long time now, glucose has been thought to be the main, if not the sole substrate for brain energy metabolism. Recent data nevertheless suggest that other molecules, such as monocarboxylates (lactate and pyruvate mainly) could be suitable substrates. Although monocarboxylates poorly cross the blood brain barrier (BBB), such substrates could replace glucose if produced locally. The two key enzymatic systems required for the use and production of these substats are lactate dehydrogenase (LDH; EC 1.1.1.27) that catalyses the interconversion of lactate and pyruvate and the pyruvate dehydrogenase complex that irreversibly funnels pyruvate towards the mitochondrial TCA cycle and oxydative phosphorylation. Our study consisted in localizing these different systems with various histochemical procedures in the cat brain and two regions, i.e. hippocampus and primary visual cortex, of the human brain. First, by means of in situ hybridization with 33P labeled oligoprobes, we have demonstrated that the more oxidative enzymes (LDH-1 and PDHA1, the gene coding for PDHEla) are highly expressed in a variety of feline brain structures. These structures include the hippocampus, various thalamic nuclei and the pons. The cerebral cortex exhibits also a high LDH-1 and PDHAl expression. On the other hand, LDH-5 expression is poorer and more diffuse, although the hippocampus does seem to have a higher expression. These fmdings are consistent with our previous observation of the expression of LDH1 and LDH-5 in the rodent brain (Laughton et al, 2000). Real-time PCR (TagMan tm) revealed that, in various regions, LDH-1 is effectively more highly expressed than LDH-5. In a second set of experiments, monoclonal antibodies to LDH-5 and PDHeIa were applied to cryostat sections of post-mortem human hippocampus and occipital cortex. These procedures revealed not only that the two enzymes have different regional distributions, but also distinct cellular localisation. LDH-5 immunoreactivity is solely observed in astrocytes. In the occipital cortex, the white matter and layer I are immunopositive. In the hippocampus, the alveus and CA4 show LDH-5 immunoréactivity. PDHeIa has been detected, with few exceptions, only in neurons. Layer IV of the occipital cortex was most immmunoreactive. In the hippocampus, PDHela immunoreactivity is noticed in the stratum granulosum and through CA 1 to CA3 areas. The overall observations made in this study show that there is a metabolic heterogeneity in the brain and our findings support the hypothesis of an astrocyte-neuron lactate shuttle (ANLS)(Bittar et al., 1996; Magistretti & Pellerin, 1999) where astrocytes export to active neurons lactate to fuel their energy demands.

Longitudinal neurochemical modifications in the aging mouse brain measured in vivo by (1)H magnetic resonance spectroscopy.

Alterations to brain homeostasis during development are reflected in the neurochemical profile determined noninvasively by (1)H magnetic resonance spectroscopy. We determined longitudinal biochemical modifications in the cortex, hippocampus, and striatum of C57BL/6 mice aged between 3 and 24 months . The regional neurochemical profile evolution indicated that aging induces general modifications of neurotransmission processes (reduced GABA and glutamate), primary energy metabolism (altered glucose, alanine, and lactate) and turnover of lipid membranes (modification of choline-containing compounds and phosphorylethanolamine), which are all probably involved in the frequently observed age-related cognitive decline. Interestingly, the neurochemical profile was different in male and female mice, particularly in the levels of taurine that may be under the control of estrogen receptors. These neurochemical profiles constitute the basal concentrations in cortex, hippocampus, and striatum of healthy aging male and female mice.