Genome Technology Center

Magnetic Resonance Imaging and Spectroscopy for the Discovery of New Human Disease Models

Ultra High Field, Micro-Scale, In-Vivo Biochemical Study of Drosophila

Personnel

Brian H. Null

Collaborators

Corey Liu, Stanford Magnetic Resonance Lab

Steve Conolly, Berkeley

Maj Hedejus, Varian

 

This work is supported by the National Science Foundation. NSF Award ID # 0521529.

Magnetic resonance imaging (MRI) and spectroscopy (MRS) is increasingly proven as a useful tool for the study of development in living organisms and thick or opaque tissues. The advent of MRI contrast agents which can act as reporters by responding to chemical concentration and gene expression, coupled with the robustness of Drosophila as a model organism, equipped with a diverse array of powerful genetic tools, now makes real time in-vivo imaging of complex processes a potential approach for the elucidation and study of human disease models.

early pupaOur effort is among the highest-field magnetic resonance imaging being performed presently, at 18.8 Tesla. Utilizing this power, we seek to expand the capabilities of magnetic resonance methods and build on earlier genomic studies of the life cycle of the fruit fly Drosophila melanogaster, with the eventual vision being to detect neurotransmitter molecules in the living, developing fly. By coupling this spectroscopic information with the expression data of receptor proteins and previously uncharacterized genes associated with neurotransmitter signal transduction and feedback mechanisms, we would gain spatial resolution and the ability to assay, genetic and chemical perturbations to this system in vivo. By utilizing the most genetically tractable application of magnetic resonance to a model organism we hope the results of these studies will aid the development of further human disease models in flies, and subsequent advancement of biomedical research in mammal and human studies.

GABA, the Benzodiazepine Receptor and Cytochrome P450

Recent MRS techniques developed for use in small mammals allows the quantification of the presence of gamma aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system across the animal kingdom. In humans, GABA concentrations vary with age, gender, and region of the brain, and are abnormal in neuropschiatric disorders, including epilepsy, anxiety disorders, depression, and drug addiction. GABA is the natural ligand for the Drosophila homolog of the human benzodiazepine receptor. While in vivo detection of GABA in mammals has proven challenging, recently the group at NIMH (Shen, et.al.) has shown the ability to detect GABA and other molecules in vivo, but no one has applied such techniques anywhere near the extreme power and resolution that my system is capable of. With the extremely high field strength of the instrument I am using and custom hardware presently in development, it is reasonable to expect that quantification of GABA and other molecules will be feasible, and this work could easily be expanded to other small specimens such as baby mice.

The following sub-cluster, taken from the previously published genomic analysis of the Drosophila life cycle, shows that the benzodiazepine receptor has a striking transcriptional pattern during the first 20hrs of pupal development, and is highly correlated with the transcript for cytochrome P450, which has a well-studied relationship with benzodiazepines and the benzodiazepine-GABA receptor in humans. GABA is also highly intertwined in this signalling system, and not surprisingly, the diazepine family of drugs(valium, halycion, etc.) have varying effects on the human psyche that are only partially understood at best. Notice also that there are numerous uncharacterized genes present in the cluster. Clusters such as this potentially represent a rich biochemical system that is yet to be understood.

GABA_cluster

From literature regarding cytochrome P450s and Benzodiazepine/GABA receptors in mammals and insects it is hypothetically possible that array data such as this hints at a GABA-signalling system, and it is fascinating to consider that it might be possible to correlate high-throughput expression data with in vivo imaging of signaling molecules in this system. The temporally-localized enrichment of the benzodiazepine receptor transcript provides an intriguing developmental period to examine, as well as the impetus to examine with a 3 dimensional method. Thus we are imaging the fly during metamorphosis with the ultimate goal of spectroscopically finding GABA, in addition to other metabolites and molecules that may be detectable in the spectra. It would be fascinating to determine when and where these molecules are acting on the developing organism, and how they are affected by the presence of drugs and native gene products expected to be interacting with GABA.

Instrumentation and Techniques

Recent developments in magnetic resonance instrumentation and methods have brought the feasibility of detailed in-vivo spectroscopic study of Drosophila within reach. Over the past several decades, tremendous advances have been made in the capabilities of magnetic resonance methods for imaging and spectroscopic measurement of molecular composition and physiological states of living tissues in human subjects. Additionally, such methods are increasingly being developed and utilized for the study of mammalian model organisms, such as rats and mice. Adapting the methods and instrumentation used for human patients to the rodent models has been highly beneficial, but also faces certain limitations, especially for those who are striving for higher resolution and sensitivity of chemical detection living or whole-tissue samples. Mounting of the specimen and field strength limitations pose significant challenges. Simply put, the ideal specimen for high resolution in vivo spectroscopic study would be able to remain stationary for hours or days without altering its normal physiology, and while immersed in an oxygen-rich perfluorocarbon oil. The ideal instrumentation for in vivo spectroscopy must have componentry closely matched to the size of the specimen. It must subject the specimen to the highest possible magnetic field, and must also have separate, tunable fields along three axes. Those doing in vivo studies of rodents are going to increasingly greater measures to push the limits of rodent methods, including development of custom magnetic resonance hardware, specimen anaesthesia and monitoring systems, and motorized specimen holders that spin the live animal in place, utilizing the method of ‘Magic Angle Spinning’ to improve signal to noise, in lieu of immersion in the aforementioned oil, which to our knowledge has not been accomplished for a live MRI sample elsewhere. The ideal system consists of placing a very high B(0) field across the smallest possible space, with a and a very hardy, unmoving specimen. Currently, the highest field magnetic resonance instruments are NMR spectrometers, reaching field strengths of about 20 Tesla across a sample that is typically around 5mm diameter. For comparison, clinical MRI units used for human studies typically produce about one Tesla across approximately one meter. The sample dimensions of an NMR spectrometer are ideal for the study of the fruit fly, Drosophila melanogaster.

Progress and Achievements

In addition to the development of a surviveable in-vivo specimen mounting system, thus far we have demonstrated successful imaging using several diverse modes of data acquisition, essentially demonstrating that various clinical and experimental imaging modalities can be used at ultra high field and microscopic dimensions. We have also successfully administered two distinct contrast agents both by feeding and direct injection across several developmental stages while retaining viability. Administration of contrast agents produced dramatic image improvement and indicates the future utility of more sophisticated contrast agents as indicators of physiological state, such as gene expression, pH, and calcium concentration. Pursuant to advancement of our spectroscopic goals, we are developing microcoils to be used in conjunction with a new imaging instrument.

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