Download Morphological and physiological interactions between GnRH3 and

Morphological and physiological interactions between GnRH3 and hypocretin/orexin
neuronal systems in zebrafish
Yali Zhao1 Chanpreet Singh2, David Prober2 and Nancy Wayne1
1. Department of Physiology, David Geffen School of Medicine at University of
California-Los Angeles, Los Angeles, CA 90095, USA
2. Division of Biology, California Institute of Technology, Pasadena, CA, 91125,
USA
ABSTRACT
Hypocretin/orexin (Hcrt) is a neuropeptide produced in the dorsal and lateral
hypothalamus, and is best known for regulating arousal and food intake. Recent evidence
indicates that it is also involved in reproduction as an inhibitor of hypothalamic
gonadotropin-releasing hormone (GnRH) neurons. However, little work has been done on
the anatomical and functional relationships between these two neuronal circuits. To
address this question, in this present study, we employed a variety of experimental
approaches including confocal imaging, immunohistochemistry, and electrophysiology in
transgenic zebrafish in which fluorescent proteins are genetically expressed in GnRH3
and Hcrt neurons. First, we generated a dual transgenic zebrafish line
GnRH3:EMD/Hcrt:RFP in which GnRH3 and Hcrt neurons are genetically labeled with
green and red fluorescent proteins, respectively. Using confocal microscopy, this animal
model allows us to study the interactions of these two neuronal systems from larval stage
(8 days post-fertilization, dpf) in vivo, as well as in the excised adult brain. We observed
that both Hcrt and GnRH3 neurons are expressed in the hypothalamus with dense
neuronal projections throughout the brain with close apposition between these two
neuronal circuits. Second, using immunochemistry, we found that the Hcrt receptor
(HcrtR) was expressed in hypothalamic GnRH3 neurons of both larvae and adult
zebrafish, indicating that Hcrt can directly regulate GnRH3 neurons. To elucidate the role
of Hcrt in regulating GnRH3 neuron activity, we recorded the electrical activities of
GnRH3 neurons using whole-cell and loose-patch electrophysiology and studied the
responses to Hcrt application with and without the Hcrt receptor antagonist almorexant.
Similar to findings with hypothalamic GnRH neurons in mice, Hcrt significantly
inhibited the action potential firing frequency of GnRH3 neurons in adult zebrafish, and
this effect was abolished by almorexant. To confirm this finding, we further employed
Hcrt receptor mutants (Hcrt+/- and HcrtR-/-) GnRH3:EMD transgenic adult zebrafish to
test the electrical response of the hypothalamic GnRH3:EMD neurons to the Hcrt
treatments. Hcrt failed to inhibit GnRH3:EMD neurons activity in the homozygous null
mutant (HcrtR-/-), but not in the control heterozygous mutant (HcrtR+/-) zebrafish.
Together, our findings demonstrate a close anatomical and functional relationship
between Hcrt and GnRH neuronal systems. It is the first demonstration of a link between
neural circuits controlling sleeping/arousal/feeding and reproduction in zebrafish – an
important animal model for investigating the molecular genetics of development.
Keywords: hypocretin; orexin; GnRH; development; teleost; electrophysiology
Source of research support: UCLA Office of the Vice Chancellor for Research (NW),
UCLA Office of the Dean of the School of Medicine (NW), National Institutes of Health
(DP)