Magnetic Resonance Imaging and Spectroscopy for the Discovery of New Human Disease Models; Ultra High Field, Micro-Scale, In-Vivo Biochemical Study of Drosophila
Brian H. Null, Ph.D.
This work is currently supported by the National Science Foundation.
NSF Award ID # 0521529
(Note: The links to fly MRI movies have been moved to the bottom of this page)
_____
_____Currently magnetic resonance imaging (MRI) and spectroscopy (MRS) is swiftly being proven as an important 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 Drosophilaas a model organism equipped with a diverse array of powerful genetic tools, now makes real time in-vivo imaging of complex processes a highly feasible approach for the elucidation and study of human disease models.
.
My effort represents the highest-field magnetic resonance imaging being performed presently, at 19 Tesla. Utilizing this power, I seek to expand the capabilities of magnetic resonance methods and build on my earlier genomic studies of the life cycle of the fruit fly Drosophila melanogasterto quantify over time the action of 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, I will gain spatial resolution and the ability to assay, in vivo and over time, genetic and chemical perturbations to this system. Using the smallest scale and most genetically tractable application of magnetic resonance to a model organism I predict that the results of these studies will aid the development of further human disease models in flies, and be directly applicable to the advancement of biomedical research in mammal and human studies.
.
GABA, the Benzodiazepine Receptor and Cytochrome P450
An MRS technique being developed at NIMH for use in small mammals allows the quantification of the presence of gammaaminobutyric 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 my 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 assembled and understood.
.
.
From literature regarding cytochrome P450s and Benzodiazepine/GABA receptors in mammals and insects it is clear that GABA has an important role in the hypothetical system suggested by the array data. Therefore, I am imaging the fly during metamorphosis and attempting to spectroscopically detect GABA, in addition to other metabolites and molecules that are detectable in the spectra. The presence of the benzodiazepine receptor provides the basis for the developmental period to examine, as well as the impetus to examine with a 3 dimensional method. It will 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 a method called ‘Magic Angle Spinning’ to improve signal to noise, in lieu of immersion in the aforementioned oil, which to my knowledge has not been accomplished for a live MRI sample, except for my own work. Thus the ideal system consists of placing the maximum possible field across the smallest possible space, and a very hardy, unmoving specimen. Currently, the highest field magnetic resonance instruments are NMR spectrometers, recently reaching field strengths of about 20 Tesla across a 5mm diameter sample, a phenomenonally large increase over the “high field” MRI units used for human studies producing one Tesla across approximately one meter. Thus the relative field strength to physical dimension is approximately 10,000 times that of a traditional clinical MRI. For this work I use one of the most powerful magnetic resonance devices available, and the sample dimensions of this machine 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 I 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. Further I have 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 sophisticated contrast agents as indicators of physiological state, such as gene expression, pH, and calcium concentration. Pursuant to advancement of my spectroscopic goals, I have support from a vital collaborator to construct a microcoil apparatus to be used in conjunction with my existing instrumentation.
.
.
Movie 1: Late Pupa in Cocoon (Drosophila melanogaster)
.
Movie 2: Adult Fly (Drosophila bifurca)
.
.
Previous Project: DNA microarray time course of Drosophila development
I developed a custom microarray fabrication system at the Stanford Genome Technology Center, and used these arrays to perform a variety of experiments, including hybridizations of RNA samples across entire Fly life cycle.
.
Current Collaborators:
Corey Liu, Stanford Magnetic Resonance Lab
Steve Conolly, Berkeley
Maj Hedejus, Varian
.
.
.
Brian Null, Ph.D.
Clark Center, SGTC
Stanford University
Stanford, CA 94305
E-Mail bnull@stanford.edu