Imaging Glutamate in the Human Brain at Ultra-High Magnetic Field: Advances and Applications

Glutamate is one of the primary neurotransmitters in the human brain, and many unanswered questions in neuroscience, psychiatry and medicine revolve around this molecule: its production, transport, conversion or degradation, regulation and effects. Yet, to date, methods for actually measuring glutamate within the human body are extremely limited. Amongst the few options in the medical imaging toolbox are magnetic resonance spectroscopy (MRS) and a recently introduced specialized form of magnetic resonance imaging (MRI) known as glutamate-weighted chemical exchange saturation transfer imaging, or gluCEST. MRS, while providing good specificity at high field strengths, lacks spatial or temporal resolution. GluCEST has the potential to provide excellent spatial resolution, but has generally been limited to single-slice acquisitions with sub-optimal B1 correction, precluding its wider application to volumetric measurements of brain structures. In this thesis, we present a novel way to correct gluCEST for B1 inhomogeneity, yielding higher quality images. We then demonstrate expansion of single-slice gluCEST imaging to volumetric ‘slab’ imaging, greatly expanding our ability to capture specific structures within the brain. We apply gluCEST in both two and three dimensions to investigate healthy brain physiology as well as the response of healthy subjects to transcranial magnetic stimulation (TMS). We were able to detect elevated gluCEST in the dentate gyrus in the brains of healthy subjects, the first non invasive measurement of its kind pertaining to this small but vital structure. We also detected, for the first time, a change in glutamate concentration in the brains of subjects who have received TMS. Finally, we present work in the area of spectroscopy, presenting a technique in which –in sharp contrast to existing methodologies requiring non-standard hardware-- metabolic dynamics of glutamate can be detected using only proton-based chemical shift imaging (CSI) in conjunction with oral ingestion of deuterium labeled glucose. While itself limited in spatial resolution, this ability to detect and visualize the dynamic neural metabolism of glucose to glutamate provides a deeply complimentary source of information to gluCEST. In the future, qCSI and gluCEST could be used in tandem to provide next-generation precision diagnostics for patients suffering from neurological maladies of metabolic origin

Imaging Glutamate In The Human Brain At Ultra-High Magnetic Field: Advances And Applications

Glutamate is one of the primary neurotransmitters in the human brain, and many unanswered questions in neuroscience, psychiatry and medicine revolve around this molecule: its production, transport, conversion or degradation, regulation and effects. Yet, to date, methods for actually measuring glutamate within the human body are extremely limited. Amongst the few options in the medical imaging toolbox are magnetic resonance spectroscopy (MRS) and a recently introduced specialized form of magnetic resonance imaging (MRI) known as glutamate-weighted chemical exchange saturation transfer imaging, or gluCEST. MRS, while providing good specificity at high field strengths, lacks spatial or temporal resolution. GluCEST has the potential to provide excellent spatial resolution, but has generally been limited to single-slice acquisitions with sub-optimal B1 correction, precluding its wider application to volumetric measurements of brain structures. In this thesis, we present a novel way to correct gluCEST for B1 inhomogeneity, yielding higher quality images. We then demonstrate expansion of single-slice gluCEST imaging to volumetric ‘slab’ imaging, greatly expanding our ability to capture specific structures within the brain. We apply gluCEST in both two and three dimensions to investigate healthy brain physiology as well as the response of healthy subjects to transcranial magnetic stimulation (TMS). We were able to detect elevated gluCEST in the dentate gyrus in the brains of healthy subjects, the first non invasive measurement of its kind pertaining to this small but vital structure. We also detected, for the first time, a change in glutamate concentration in the brains of subjects who have received TMS. Finally, we present work in the area of spectroscopy, presenting a technique in which –in sharp contrast to existing methodologies requiring non-standard hardware-- metabolic dynamics of glutamate can be detected using only proton-based chemical shift imaging (CSI) in conjunction with oral ingestion of deuterium labeled glucose. While itself limited in spatial resolution, this ability to detect and visualize the dynamic neural metabolism of glucose to glutamate provides a deeply complimentary source of information to gluCEST. In the future, qCSI and gluCEST could be used in tandem to provide next-generation precision diagnostics for patients suffering from neurological maladies of metabolic origin

Membrane Affinity of Platensimycin and Its Dialkylamine Analogs

Membrane permeability is a desired property in drug design, but there have been difficulties in quantifying the direct drug partitioning into native membranes. Platensimycin (PL) is a new promising antibiotic whose biosynthetic production is costly. Six dialkylamine analogs of PL were synthesized with identical pharmacophores but different side chains; five of them were found inactive. To address the possibility that their activity is limited by the permeation step, we calculated polarity, measured surface activity and the ability to insert into the phospholipid monolayers. The partitioning of PL and the analogs into the cytoplasmic membrane of E. coli was assessed by activation curve shifts of a re-engineered mechanosensitive channel, MscS, in patch-clamp experiments. Despite predicted differences in polarity, the affinities to lipid monolayers and native membranes were comparable for most of the analogs. For PL and the di-myrtenyl analog QD-11, both carrying bulky sidechains, the affinity for the native membrane was lower than for monolayers (half-membranes), signifying that intercalation must overcome the lateral pressure of the bilayer. We conclude that the biological activity among the studied PL analogs is unlikely to be limited by their membrane permeability. We also discuss the capacity of endogenous tension-activated channels to detect asymmetric partitioning of exogenous substances into the native bacterial membrane and the different contributions to the thermodynamic force which drives permeation

Dataset for: The thermodynamic basis of glucose stimulated insulin release: a model of the core mechanism.

A model for glucose sensing by pancreatic β-cells is developed and compared to the available experimental data. The model brings together mathematical representations for the activities of the glucose sensor, glucokinase, and oxidative phosphorylation. Glucokinase produces glucose 6-phosphate (G-6-P) in an irreversible reaction that determines glycolytic flux. The primary products of glycolysis are NADH and pyruvate. The NADH is reoxidized and the reducing equivalents transferred to oxidative phosphorylation by the glycerol phosphate shuttle and some of the pyruvate is oxidized by pyruvate dehydrogenase and enters the citric acid cycle. These reactions are irreversible, and result in a glucose concentration dependent reduction of the intramitochondrial NAD pool. This increases the electrochemical energy coupled to ATP synthesis, and thereby the cellular energy state ([ATP]/[ADP][Pi]). [ATP] and [Pi] are 10 to 100 times greater than [ADP], so the increase in energy state is primarily through decrease in [ADP]. The decrease in [ADP] is considered responsible for altering ion channel conductance and releasing insulin. Applied to the reported glucose concentration dependent release of insulin by perifused islet preparations (4), the model predicts that the dependence of insulin release on [ADP] is strongly cooperative with a threshold of about 30 µM and a negative Hill coefficient near -5.5. The predicted cellular energy state, [ADP], creatine phosphate/creatine ratio, and cytochrome c reduction, including their dependence on glucose concentration, are consistent with experimental data. The ability of the model to predict behavior consistent with experiment is an invaluable resource for understanding glucose sensing and planning experiments

Membrane Affinity of Platensimycin and Its Dialkylamine Analogs

Membrane permeability is a desired property in drug design, but there have been difficulties in quantifying the direct drug partitioning into native membranes. Platensimycin (PL) is a new promising antibiotic whose biosynthetic production is costly. Six dialkylamine analogs of PL were synthesized with identical pharmacophores but different side chains; five of them were found inactive. To address the possibility that their activity is limited by the permeation step, we calculated polarity, measured surface activity and the ability to insert into the phospholipid monolayers. The partitioning of PL and the analogs into the cytoplasmic membrane of E. coli was assessed by activation curve shifts of a re-engineered mechanosensitive channel, MscS, in patch-clamp experiments. Despite predicted differences in polarity, the affinities to lipid monolayers and native membranes were comparable for most of the analogs. For PL and the di-myrtenyl analog QD-11, both carrying bulky sidechains, the affinity for the native membrane was lower than for monolayers (half-membranes), signifying that intercalation must overcome the lateral pressure of the bilayer. We conclude that the biological activity among the studied PL analogs is unlikely to be limited by their membrane permeability. We also discuss the capacity of endogenous tension-activated channels to detect asymmetric partitioning of exogenous substances into the native bacterial membrane and the different contributions to the thermodynamic force which drives permeation