Excitatory/inhibitory neuronal metabolic balance in mouse hippocampus upon infusion of [U-C-13(6)]glucose

Hippocampus plays a critical role in linking brain energetics and behavior typically associated to stress exposure. In this study, we aimed to simultaneously assess excitatory and inhibitory neuronal metabolism in mouse hippocampus in vivo by applying (18)FDG-PET and indirect C-13 magnetic resonance spectroscopy (H-1-[C-13]-MRS) at 14.1 T upon infusion of uniformly C-13-labeled glucose ([U-C-13(6)]Glc). Improving the spectral fitting by taking into account variable decoupling efficiencies of [U-C-13(6)]Glc and refining the compartmentalized model by including two gamma-aminobutyric acid (GABA) pools permit us to evaluate the relative contributions of glutamatergic and GABAergic metabolism to total hippocampal neuroenergetics. We report that GABAergic activity accounts for similar to 13% of total neurotransmission (V-NT) and similar to 27% of total neuronal TCA cycle (V-TCA) in mouse hippocampus suggesting a higher V-TCA/V-NT ratio for inhibitory neurons compared to excitatory neurons. Finally, our results provide new strategies and tools for bringing forward the developments and applications of C-13-MRS in specific brain regions of small animals.

Mathematical Modeling of Brain Energy Metabolism, Measured with PET and MRS in Rodents

Bernard Lanz

Positron emission tomography (PET) and nuclear magnetic resonance spectroscopy (MRS) are two biomedical measurement techniques developed in the end of the XXth century, which drastically improved the amount of accessible information available in vivo. PET became popular through the most widely used tracer, fluorodeoxyglucose (FDG), which enables the measurement of the local glucose utilization and is nowadays routinely applied in clinical practice. Nuclear magnetic resonance is used in a large array of applications such as in analytical chemistry or chemical structure determination and is essentially known for its versatile medical imaging capabilities, grouped under the name of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). In vivo MRS measures concentrations of metabolites by interacting with various nuclei such as 1H, 13C or 15N, with the major asset that MRS enables the identification of the chemical position at which the detected nuclei is located in the measured molecule. When coupled with labeled substrate infusions, dynamic MRS gives the opportunity to probe specific biochemical reactions directly in vivo, which opens the way to a wide range of metabolic studies. However, the correct interpretation of the elaborate dynamic labeling data acquired either with MRS or PET tracer experiments requires the application of adapted metabolic models to derive quantitative metabolic rates characterizing the biochemical processes under study. The work of this thesis involves both PET and MRS studies of brain energy metabolism in rodents and focuses on the development of adapted metabolic models to derive reliable metabolic fluxes characterizing various brain metabolic processes, such as glucose consumption, glial and neuronal oxidative metabolism and neurotransmission. FDG-PET enables the measurement of the cerebral metabolic rate of glucose (CMRGlc) using a three-compartment metabolic model for FDG transport across the blood-brain barrier and FDG phosphorylation to FDG-6P. Although this method is well established, a main drawback of CMRGlc measurement with FDG-PET is the necessity to characterize the available FDG concentration in the plasma over the experiment duration, the so-called arterial input function. This constraint is a strong limitation for preclinical studies in rodents, due to their low blood volume. We developed therefore a new method to extract the FDG input function directly from the PET images in rats and mice, using the time-activity curve of a voxel located in the inferior vena cava. The method was validated by comparison with a dedicated external blood counter and manual blood sampling. Using this method, a CMRGlc of ~0.22 μmol/g/min was determined in the mouse cortex. Glial oxidative metabolism can be specifically assessed using glial specific substrates, in particular [1-11C] acetate. However, no existing metabolic model described the 11C tissue activity curve in terms of neuroglial energy metabolism. In order to extract quantitative metabolic fluxes characterizing the glial TCA cycle rate and glutamate/glutamine cycling, we adapted the neuroglial two-compartment modeling approach previously used in 13C MRS cerebral metabolic studies to interpret positron emission data following 11C-acetate injection in the rat. The precision and accuracy of the estimated metabolic parameters was tested and a composite glial metabolic flux Vgtg=0.136 ± 0.042 μmol/g/min and a neurotransmission flux Vnt=0.170 ± 0.103 μmol/g/min were obtained. This approach enabled a direct comparison of the metabolic fluxes measured using 11C PET with the values determined with 13C MRS. In the field of 13C MRS, we took advantage of the higher sensitivity and spectral resolution available at high magnetic field (14.1T) to measure glial and neuronal oxidative metabolism, glutamatergic neurotransmission and pyruvate carboxylation in more details and with higher precision using both [1,6-13C2] glucose and [2-13C] acetate infusions in rats. In the acetate study, the glial specific uptake of acetate enabled a quantification of glial oxidative metabolism with unrivalled precision. We could estimate for the first time separately the transmitochondrial flux Vx in the glial and neuronal compartment, a biochemical pathway which has been subject of strong controversy. The glial and neuronal Vx were determined with high precision, as calculated through Monte Carlo simulation, and their value was on the same order of magnitude than the respective glial and neuronal TCA cycle flux. The model was extended to estimate the fraction of glutamate located in the glial compartment. A glial glutamate concentration of 0.6 ± 0.1 μmol/g was found. The values of the different metabolic fluxes characterizing the neuroglial system (Vtcag=0.27, Vxg=0.17, VPC=0.087, VNT=0.15, Vtcan=0.37 and Vxn=0.46 μmol/g/min) were in very good agreement with the values found using [1,6-13C2] glucose infusion. In addition, the effect of the MRS temporal resolution, experiment duration and signal- to-noise ratio on the precision of the derived metabolic fluxes was analyzed. Previous simulation studies showed that two-compartment modeling of neuroglial metabolism using 13C glucose infusion could lead to unreliable parameter estimations. However, using polarization transfer 13C MRS at 14.1T, the carbon positions C4, C3 and C2 of glutamate and glutamine as well as the positions C3 and C2 of aspartate could be measured with high SNR. The C2 positions were used in two-compartment modeling for the first time, enabling a precise measurement of the apparent glutamatergic neurotransmission and glial metabolism, including pyruvate carboxylation, as confirmed by Monte Carlo simulation. The glial dilution at the level of acetyl-CoA, which is related to the metabolism of other substrates than glucose, could also be characterized. Brain metabolism under hyperammonemic conditions was probed with 15N MRS under labeled ammonia infusion and modeled using a modified neuroglial two- compartment model describing the dynamics of [5-15N] glutamine, [2-15N] glutamate and [2-15N] glutamine. Due to detoxification processes, the total glutamine concentration, measured with 1H MRS, was steadily increasing. This non-steady- state metabolic condition resulting from the hyperammonemic stress was taken into account in the model, resulting in the determination of the apparent neurotransmission (0.26 ± 0.030 μmol/g/min), the glutamate dehydrogenase (0.029 ± 0.002 μmol/g/min) and net glutamine accumulation (0.033 ± 0.001 μmol/g/min). Finally, compartmental modeling was applied to 13C labeling studies in the awake rat, fed during 5, 24 or 48 h with a [1-13C]-labeled glucose solution, to determine brain glycogen and NAA turnover times (τGlyc=5.3 ± 3.2 h and τNAA=15.6 ± 6.5 h), using 13C spectra of brain extracts. A group of rats followed a mild brain activation protocol over 5 hours, which resulted in a decreased glycogen turnover time (τGlyc =2.9 ± 1.2 h).

EPFL2012

Elucidating the role of neuron-astrocyte metabolic coupling in learning and memory

Monika Tadi

The brain has very high energy demands that are mainly met by the circulating blood glucose to ensure its proper functioning. Thus, it is not surprising that though the human brain weighs only 2- 3% of the body weight, it consumes approximately 25% of total body glucose. “Neuro-energetic coupling" is a unique feature of the brain and refers to the existence of the tight coupling between (neuronal) synaptic brain activity and energy metabolism. The main excitatory neurotransmitter in the brain is glutamate and most of the energy requirements at the cortical level are met through glutamate-mediated neurotransmission, suggesting that there is a close relationship between brain activity, glutamatergic neurotransmission and energy metabolism. Until the 80s, blood-borne glucose was thought to be the sole energy substrate of the brain cells and it was assumed that glucose is metabolized by neurons and astrocytes (a type of glial cell) independently. It is only during the last two decades that the concept of compartmentalization of energy metabolism between neurons and astrocytes has become more obvious. The transfer of lactate from astrocytes to neurons termed as the “astrocyte-neuron lactate shuttle (ANLS)” model proposed by Dr. Pellerin and Prof. Magistretti posits that glutamate released during brain activation activates glycolysis (one of glucose metabolic pathways) in astrocytes leading to lactate (a monocarboxylate) release in the extracellular space, and this astrocytic lactate is then captured and used as an energy substrate by the activated neurons to meet their energy requirements. It includes a complex chain of events involved in neuro-metabolic coupling and supports the preferential use of lactate by activated neurons under conditions of increased energy demands. Higher brain functions such as learning a new task are associated with structural changes in brain and are metabolically demanding necessitating the need of an additional energy supply to meet the neurons increased energy requirement. In this context, the current thesis project aimed to elucidate the contribution of metabolic coupling between astrocytes and neurons, known as “neuron-astrocyte metabolic coupling” to learning and memory formation. Using an in vivo approach combined with behavioral, molecular and gene manipulation techniques in mice, we set out to investigate the role of neuron-astrocyte metabolic coupling, particularly ANLS in cognition. The project was divided into two main parts: In the first part, the expression of glucose metabolism-related genes was investigated during long term memory (LTM) formation in fear-motivated inhibitory avoidance (IA) learning paradigm. Using (14C) 2-Deoxyglucose (2-DG) technique, we first defined the brain areas that are involved in IA LTM formation. This technique provides an indirect measurement of brain activity by measuring glucose uptake in different brain areas during the task. The results of brain metabolic mapping show an increase in glucose utilization in the hippocampus, amygdala, anterior cingulate cortex and pre-limbic and infra-limbic cortical areas. Results from the quantitative analysis of mRNA levels in the dorsal hippocampus at different time point’s following IA task highlight a time dependent, learning specific change in the expression of genes related to glucose metabolism. Specific set of genes involved in the synthesis and degradation of glycogen (brain glucose reserve present only in astrocytes), pyruvate metabolism and pentose phosphate shunt as well as the ANLS were induced following the IA task. These early observations indicate that the metabolic interactions between astrocytes and neurons undergo modifications during a learning process and suggest that these adaptations may play a key role in the establishment of long-term memory. In the second part of research project, we focused on investigating the behavioral consequences of down regulating the expression of Monocarboxylate Transporter 1 (MCT1), a key ANLS related gene, on cognition and higher brain functions. Mice heterozygous for the MCT1 gene (MCT1+/-) were characterized in numerous behavioral paradigms such as general wellbeing, reflexive and motor capabilities. After having established that MCT1+/- mice have no alterations in general emotional state or in locomotion, a battery of behavioral tests was performed to study cognition in these mice. Compared with wild-type control mice, the MCT1+/- mice display normal muscle strength and motor coordination. However, when tested for higher brain functions, the MCT1+/- mice exhibit learning and memory deficits in several learning paradigms/tasks that are hippocampus and / or amygdala dependent. The cognitive impairment observed in the MCT1 heterozygous mice further highlights the importance of ANLS in memory and suggest that disruption of lactate transfer from astrocytes to neurons via the MCT1 results in cognitive impairment. The overall results in this thesis demonstrate that cerebral energy metabolism, and in particular the transfer of lactate from astrocytes to neurons not only provides temporal adaptations in the learning process, but it also plays a fundamental role in memory formation.

EPFL2013

In vivo magnetic resonance studies of glycogen and hyperpolarized lithium in the rat brain

Ruud van Heeswijk

Nuclear magnetic resonance (NMR) is used for a large array of applications, ranging from chemical characterization to oil drilling to medical imaging. In these fields NMR is used as an investigational tool, but new techniques and applications are continuously being developed as well. The latter is also the subject of this thesis. After an introduction to NMR theory, it focuses on the development of new methodologies for two separate areas of investigation: the metabolism of brain glycogen and hyperpolarization via dynamic nuclear polarization (DNP). Brain glycogen is the main storage form of glucose in the brain. However, because it is present in much lower concentrations in the brain than in other tissues, the significance of this storage and other roles are subject to debate. To better understand the roles of brain glycogen, it is important to quantify its metabolic parameters. The only currently available method to detect brain glycogen in vivo is 13C NMR spectroscopy. Incorporation of 13C-labeled glucose is necessary to allow glycogen measurement, but might be affected by changes in the turnover between glucose and glycogen. We therefore established a protocol to measure the glycogen absolute concentration in the rat brain by eliminating label turnover as a variable. The approach is based on establishing an increased, constant 13C isotopic enrichment (IE). 13C-glucose infusion is then performed at this IE of brain glycogen. As glycogen IE cannot be assessed in vivo, we validated that it can be inferred from the N-acetyl-aspartate (NAA) IE in vivo: after [1-13C]-glucose ingestion, glycogen IE was 2.2 ± 0.1 times that of NAA. Glycogen concentration measured in vivo by 13C NMR (5.8 ± 0.7 µmol/g) was in excellent agreement with post-mortem biochemistry (6.4 ± 0.6 µmol/g). A second glycogen NMR protocol was then implemented to measure concentration and turnover simultaneously. After reaching isotopic steady state for glycogen C1 using [1-13C]-glucose administration, [1,6-13C2]-glucose was infused such that isotopic steady state was maintained at the C1 position, but the C6 position reflected 13C label incorporation. To overcome the large chemical shift displacement error between the C1 and C6 resonances of glycogen, 2D gradient-based localization using the Fourier series window approach was implemented. The glycogen concentration of 5.1 ± 1.6 µmol/g measured from the C1 position was in excellent agreement with concomitant biochemical determinations, while glycogen turnover measured from the rate of label incorporation into the C6 position of glycogen in the α-chloralose anesthetized rat was 0.7 µmol/g/h. The contrast agent Omniscan was added to a formic acid-filled glass reference sphere in the abovementioned protocols in order to shorten its T1 relaxation time and to accelerate the calibration procedure it is used in; therefore it was established that Omniscan in pure 13C formic acid has a relaxivity of 2.9 mM-1s-1. The study was expanded, and the relaxivity of several commercially available gadolinium (Gd)-based contrast agents was determined for X-nuclei resonances with long intrinsic relaxation times. The relaxivity of Omniscan on the glutamate C1 and C5 in aqueous solution was ~ 0.5 mM-1s-1. These relaxivities allow the preparation of solutions with a predetermined short 13C T1. Another isotope with a long relaxation time is lithium-6 (6Li). Lithium is an ion that is widely used in psychotherapy, and 6Li has an intrinsic relaxation time on the order of several minutes, which was strongly affected by the contrast agents. Its relaxivity ranged from ~ 0.1 mM-1s-1 for Omniscan to 0.3 for Magnevist, whereas the relaxivity of Gd-DOTP was 11 mM-1s-1. Overall, these experiments suggested that the presence of submicromolar contrast agent concentrations should be detectable, provided sufficient sensitivity is available, such as that afforded by hyperpolarization. This submicromolar contrast agent concentration detection was demonstrated in a subsequent study: first it was established that lithium-6 can be readily hyperpolarized through DNP within 30 min while retaining a long polarization relaxation time on the order of a minute. The shortening of this relaxation time was used for the detection of 500 nM contrast agent in vitro. Hyperpolarized lithium-6 was then administered to the rat, where its signal retained a relaxation time on the order of 70 s in vivo. Localization experiments implied that the lithium signal originated from within the brain and that it was detectable up to 5 min after administration. Next, ~ 1.5 µM of contrast agent was injected in the rat, and its effect on previously injected hyperpolarized lithium-6 was successfully quantified. It was concluded that the detection of submicromolar contrast agent concentrations using hyperpolarized NMR nuclei such as 6Li may provide a novel avenue for molecular imaging. In a separate set of of experiments, hyperpolarization through DNP was applied to 15N-labeled choline, 13C-labeled bicarbonate and 13C-labeled acetate. All three metabolites were successfully polarized and were detected both in vitro and in vivo.

EPFL2009