Molecular Imaging of Compartmentalized Cerebral Carbohydrate Metabolism by In Vivo Multiphoton Microscopy of NADH Fluctuations in Cortical Astrocytes and Neurons

Karl A. Kasischke, M.D.

University of Rochester Medical Center, Rochester, NY

Grant Program:

David Mahoney Neuroimaging Program

Funded in:

December 2006, for 3 years

Funding Amount:

$300,000

Lay Summary

Cellular Imaging May Provide Clues to Metabolic Disturbances in Several Brain Diseases

This laboratory animal study will determine whether two-photon imaging can discern how metabolic processes that create energy for brain cells are altered by degenerative diseases and injuries.

The brain depends on a steady supply of glucose (sugar) and oxygen to satisfy its high energy demands. This supply can be dangerously interrupted by stroke, diabetes, or degenerative diseases. Yet current imaging techniques, such as MRS, do not enable researchers to directly identify metabolic alterations that could be targeted by experimental drug therapies. The researchers, therefore, will determine whether two-photon microscopy’s high resolution can track a naturally fluorescent co-enzyme (NADH) that is involved in the metabolism of oxygen and glucose, to precisely localize metabolic fluxes in the brains of laboratory animals.

They anticipate this imaging approach will demonstrate that “neurometabolic coupling” occurs. Specifically, they hypothesize, neurons metabolize oxygen, and then astrocytes become activated and break down glucose to release energy that sustains this metabolic process in neurons. If two-photon imaging effectively lays the groundwork for quantifying neural energy metabolism in the intact brain, it may help clinical researchers interpret MRS imaging of brain metabolism in various human diseases.

Significance: Two-photon NADH imaging in animals may be able to address fundamental questions concerning cellular energy metabolism abnormalities involved in neurodengerative and metabolic brain diseases. This could help inform interpretation of MRS findings in people with these diseases and lead to development of new therapeutic approaches.

Hypothesis

Hypothesis: I hypothesize that two-photon NADH imaging and spectroscopy is a novel neuroimaging modality to directly localize and quantify compartmentalized oxidative and glycolytic glucose metabolism in neurons and astrocytes and their processes in the intact, living, and activated rodent brain.

Goals: The goal of my research is to develop and define two-photon in vivo NADH imaging/spectroscopy as a novel microscopic functional imaging modality with the unique capabilities to resolve activity-dependent metabolism in single neural cells and their processes in the intact, living rodent brain. I address unresolved questions which are fundamental to our understanding of brain metabolism and function. 1. How is activity-dependent oxidative and glycolytic metabolism divided between neurons and astrocytes? 2. What are the coupling mechanisms leading to specific activation of astrocytic energy metabolism? 3. What is the exact relationship between vascular architecture and blood flow and the metabolic state of the brain tissue within the capillary bed in the rodent barrel cortex?

Methods: Developments in multiphoton imaging technology include novel optical filters and lenses, scanning devices, fiber–coupled spectrometers, photomultiplier tubes, and image processing. Two-photon NADH imaging will be performed in the rodent barrel cortex following whisker stimulation. Cranial window surgery with mechanical ventilation and blood gas monitoring, and electrical recordings of brain activity and oxygen potential will be performed. Identification of astrocytes and neurons and the microvasculature will be achieved by using transgenic mice, external fluorophores and microangiographies.

Selected Publications

Kasischke K.A. A new pathway for lactate production in the CNS. J Physiol. 2008 Mar 1;586(5):1207-8.

Takano T., Tian G.F., Peng W., Lou N., Lovatt D., Hansen A.J., Kasischke K.A., and Nedergaard M. Cortical spreading depression causes and coincides with tissue hypoxia. Nat Neurosci. 2007 Jun;10(6):754-62.

Norman J.P., Perry S.W., Kasischke K.A., Volsky D.J., and Gelbard H.A. HIV-1 trans activator of transcription protein elicits mitochondrial hyperpolarization and respiratory deficit, with dysregulation of complex IV and nicotinamide adenine dinucleotide homeostasis in cortical neurons. J Immunol. 2007 Jan 15;178(2):869-76.

Vishwasrao H.D., Heikal A.A., Kasischke K.A., Webb W.W. Conformational dependence of intracellular NADH on metabolic state revealed by fluorescence associated anisotropy. (Cover article) J Biol Chem. 2005 Jul 1;280(26):25119-26.

Kasischke K.A., Vishwasrao H.D., Fisher P.J., Zipfel W.R., and Webb W.W. Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science. 2004 Jul 2;305(5680):99-103.