Principal Investigator
Fumihito Ono, M.D., Ph.D.
TS32, 5625 Fishers Lane, Rockville, MD. 20852
Office: 301-443-3748
Lab: 301-443-3772
Fax: 301-480-0466
email: onof@mail.nih.gov

Mission Statement:

Our goal is to clarify basic principles of synapse function by using a genetic model system, zebrafish. We use mutant fish with abnormal behaviors. By introducing modified forms of neural proteins into these fish, we can change their behavior. Thanks to the rapid and external development of zebrafish, neural mutants survive long enough to allow analysis of neural functions before they die. The transparency of zebrafish embryos also enables an optical observation: in vivo observation of genetically marked neural proteins, in particular. These unique features of zebrafish present a valuable opportunity to study the developing nervous system in vivo.


Current Projects:


Zebrafish as a model to study nervous system 

The section on model synaptic systems started in May 2007, when Fumihito Ono moved to NIH/NIAAA from the University of Florida. Though projects have undergone some shifts after moving to NIAAA, the research in our lab has always focused on the nervous system. In particular, we are interested in how neural functions lead to various behaviors of animals. We use a model system, zebrafish, to address this question. Zebrafish offer unique advantages that complement other commonly-used model systems such as mice.

We are currently studying the nervous system at several levels. An experimental paradigm we have used heavily for the past several years is the neuromuscular junction (NMJ). NMJ is a synapse between a motor neuron and a muscle cell. Not only is this synapse directly linked to various diseases arising from genetic defects, it also offers an exceptional accessibility for an array of experimental techniques. As a result, NMJ is the best-studied synapse in vertebrate biology. We have recently broadened our field and are now studying neural networks in the central nervous system and its response to ethanol.


Projects using neuromuscular junction

Our projects on NMJ center around locomotory mutants we discovered to have defects in two key molecules of the neuromuscular synapse. One mutant lacks acetylcholine receptors (AChR) in the muscle. As a result, the fish cannot mount a movement when the motor neuron releases ACh. Another mutant has a dysfunctional rapsyn. Rapsyn is a post-synaptic protein that brings AChRs together. In this fish, AChRs do not make clusters at the synapse and are diffusely distributed over the muscle cell surface. From the AChR-less mutant, we found that AChR plays an active role, directing rapsyn molecules to synapse. In rapsyn mutant fish, we found that AChRs not only fail to form clusters at synapse, but also their functions are altered. When motor neurons fire at a high frequency, the amplitude of AChR current remains constant in wild type, whereas in rapsyn-mutant fish the response shows a marked attenuation with repeated firing of motor neurons. Several projects aim to figure out the mechanisms underlying these unexpected functions of AChR and rapsyn.
 
AChR-less fish has an analogous disease in humans, which is called Fetal Akinesia Deformation Sequences (FADS). Human embryos that harbor mutation in one of the AChR genes suffer premature death in the first trimester. We introduced a modified AChR gene into the mutant fish. The introduced gene expressed in all muscle cells, which led to a successful rescue of the mutant fish. The rescued fish survives well beyond sexual maturation, and they can mate normally, producing offspring. To the best of our knowledge, this is the first case of a mutant animal corresponding to first trimester lethality in human that has been rescued by a transgene and survived to adulthood. This result is reported in a paper now in press for the Journal of Neuroscience (Epley et al., 2008).


The AChR-less mutant and the rapsyn mutant also provide a unique opportunity to study functions of AChRs expressed in the central nervous system (CNS) which play important roles in various neural disorders including alcohol¬ism. In the neuromuscular synapse of AChR-less mutant, we showed that pre-synaptic machinery releasing ACh develops normally. These AChR-less mutants therefore allow us to study brain-type AChRs in a real synaptic context, when the receptors are ectopically expressed in the muscle cells. We are using this "model synapse" to study functions of brain type AChR
receptors, a4J32 and a7, which are difficult to study in their native environment.
 
Projects on the central nervous system

One of the major advantages of zebrafish is that it is amenable to large-scale forward genetics. We have performed several forward genetic screenings on zebrafish. A transgenic zebrafish isolated from one such screening expresses red fluorescent protein (RFP) in a sub-population of neurons. In these neurons, the expression of a transcription factor pax8, whose gene was “trapped” by the RFP gene, is replaced by RFP. In the spinal cord, the labeling of pax8 expressing cells in vivo enabled us to identify a population of inter-neurons, which was not previously described. We are now characterizing these inter-neurons in terms of their connections to other spinal neurons, and studying the effect of deleting pax8 and related genes for the neural network in the spinal cord. Use of this and other lines of fish will allow us to study functions of developmental genes in living animals.
 
Implications for ethanol-related diseases

The link of on-going projects in the lab to ethanol can be found at several levels. The AChRs we are expressing in the NMJ determine the sensitivity of brain to nicotine, and it is implicated that alcohol and nicotine exert reciprocal interactions in the human brain, as suggested by the co- morbidity of alcoholism and nicotine addiction.

We are also performing experiments to study the direct effects of ethanol on the zebrafish CNS, using an array of techniques we have developed over the years. We are studying the intoxicated embryos at the level of genomics, cellular physiology, anatomy and locomotion. Embryos grown in Ethanol in these experiments are comparable to fetuses with Fetal Alchol Syndrome (FAS) in human. Insights gained from these experiments will help to clarify the mechanisms of FAS.


Publications 2007-2008:

Ono F. An emerging picture of synapse formation: A balance of two opposing pathways.

Science Signaling 1: pe3, 2008.

Epley K, Urban J, Ikenaga T, Ono F. A modified acetylcholine receptor d-subunit enables a null mutant to survive beyond sexual maturation.

J Neurosci In press, 2008.

Current Staff:

Postdoctoral Fellow

Jason Urban, Ph.D.
Jee-Young Park

Postbaccalaureate Fellow

Alison Delargy, B.A.

Alumni

Postdoc
Kimberly Epley

Graduate Student
Nicole Gebhart

Undergraduate
Elizabeth Jimenez
Dana Smith
Meghan Mott
Jarrod Smith

Updated: May 2009