Ricardo Miledi

Distinguished Professor, Neurobiology and Behavior
School of Biological Sciences
Molecular Biology and Biochemistry
School of Biological Sciences

M.D., Universidad Nacional Autonoma de Mexico

Phone: (949) 824-4721, 5693
Fax: (949) 824-3522
Email: miledi@uci.edu

University of California, Irvine
1215 McGaugh Hall
Mail Code: 4550
Irvine, CA 92697
Research Interests
Synaptic physiology and molecular neurobiology; neurotransmitter receptor function
Research Abstract
The brain contains myriads of nerve cells that are linked by a bewildering number of synaptic connections. Brain function, with all its implications, depends upon the transmission of signals across synapses, many of which are chemical. That is, they operate via the presynaptic release of transmitter substances which act on receptors embedded in the membrane of the postsynaptic cell. Furthermore, the release of transmitters and the responses that they induce in postsynaptic cells depend upon the presence of voltage-activated channels in the cell membranes. The major aim of our research is to study, at cellular and molecular levels, the functioning and structure of the many receptors and channels that underlie brain functions.

To obtain a thorough understanding of the structure and function of these receptors and channels requires the applications of diverse techniques. We are combining expertise in gene cloning, electrophysiology, single channel recording, monoclonal antibodies, cell culture and in vitro formation of synapses, neurotoxins, light and electron microscopy, and monitoring of intracellular ion concentrations.

Much of our work revolves around a novel technique that allows the functional expression of receptor and channel proteins from the messenger RNAs that encode their structures. Detailed knowledge of receptors and channels in the brain is at present meager. To a large extent, this is due to the small size and inaccessibility of neurons in the brain, particularly in the human brain. It seemed to us that these problems could be avoided if it were possible to induce larger cells to acquire the receptor and channel proteins that occur in brain neurons. For this purpose, we chose the oocytes of the frog Xenopus laevis, which are over 1 mm in diameter and can easily be micro-injected with foreign messenger RNA. Following injection of brain messenger RNAs (including those from humans), the oocyte expressed receptors and channels that could be activated by application of drugs or by changes in membrane potential. In this way, we have "transplanted" many of the known brain neurotransmitter receptors to the oocyte, including receptors to acetylcholine, catecholamines, excitatory and inhibitory amino acids, serotonin, and peptides.

Once expressed in the large oocyte cells, the study of receptor function becomes much easier. For example, we use such techniques as voltage clamp, noise analysis, and patch clamp recording to examine the currents flowing through the ion channels activated by receptor binding, and to see how these currents are modified by clinically useful drugs. Another topic that becomes more amenable for study is the role of intracellular messengers, which are used in several neurotransmitter systems as the link between receptor binding and activation of membrane channels. In the oocyte, it is possible to monitor putative second messengers such as calcium and cyclic nucleotides during activation, and also to examine the effect of injection of these substances into the cell.

A different application is to use the oocyte as a screening system to test and develop new drugs, and to measure the extent to which messenger RNAs coding for different neurotransmitter receptors are expressed in different tissues. For example, messenger RNAs are being isolated from different areas of the brain, from brains at different stages or embryological development, or following various pathological conditions such as Alzheimer's disease.

One of the most important applications, however, concerns the use of the oocyte system as an aid in the cloning of the genes coding for receptors and electrically excitable channels. Injection of messenger RNAs transcribed from the cloned genes induced functional receptor/channels in the oocyte. Once clones are obtained, the DNA can be sequenced to yield information about the likely structure of the proteins. The ultimate aim is to explain the observed functioning of the receptor and channel molecules in terms of their predicted structures.
Tigyi, G., Matute, C. & Miledi, R. (1990) Monoclonal antibodies to cerebellar pinceau terminals obtained after immunization with brain mRNA-injected Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 87, 528-532
Arellano, R.O. & Miledi, R. (1994) Osmo-dependent Cl- currents activated by cyclic AMP in follicle-enclosed Xenopus oocytes. Proc. R. Soc. London B 258, 229-235.
Garcia-Colunga, J.J. & Miledi, R. (1995) Effects of serotonergic agents on neuronal nicotinic acetylcholine receptors. Proc. Natl. Acad. Sci. U.S.A. 92, 2919-2923.
Marsal, J., Tigyi, G. & Miledi, R. ((1995) Incorporation of actylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes. Proc. Natl. Acad. Sci. U.S.A. 92, 5224-5228.
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