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Octopamine receptor in mushroom bodies: Biological Overview | References

Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. This study investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, protein kinases functionally interacting with OAMB were explored by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from the ovary to the uterus (Lee, 2009).

Mating activates highly coordinated physiological processes in the Drosophila female. During copulation, the female receives somatosensory stimulation, sperm and seminal proteins from the male. These mating signals act at multiple sites in the mated female to activate post-mating responses required for successful reproduction. For example, the seminal protein Ovulin stimulates egg-laying for 1 day after mating (Herndon, 1995). Ovulin is not only present in the base of the ovary right after copulation but it also enters the circulatory system, possibly acting at additional sites. Moreover, the seminal sex peptides Acp70A and DUP99B reduce sexual receptivity and stimulate egg-laying. While sex peptides have widespread binding sites in the central nervous system, endocrine glands, and reproductive tissues in the female, it is the sex peptide receptor SPR in the neurons expressing the sex determination factor Fruitless that is indispensable for reduced receptivity as well as increased egg-laying (Lee, 2009).

The downstream targets and mechanisms that Ovulin and SPR activate in the mated female are unknown. Several studies, nonetheless, indicate octopamine as a key neuromessenger for ovulation (Lee, 2003; Monastirioti, 2003; Cole, 2005; Rodriguez-Valentin, 2006; Middleton, 2006), suggesting it being a downstream signal of Ovulin or SPR for egg-laying. Octopamine is a major monoamine in insects and has similar functions to mammalian norepinephrine. Octopamine is synthesized from tyrosine by sequential actions of tyrosine decarboxylase (dTdc) and tyramine beta-hydroxylase (Tβh). The females defective in dTdc2 encoding neuronal dTdc or tβh are sterile due to defective egg-laying (Monastirioti, 2003; Cole, 2005). Octopaminergic neurons innervate numerous brain and thoracico-abdominal ganglion (TAG) areas (Monastirioti, 1995; Sinakevitch, 2006). In addition, octopaminergic neurons in the TAG project to reproductive tissues such as the ovaries, oviducts, sperm storage organs and uterus (Rodriguez-Valentin, 2006; Middleton, 2006). Indeed, the sterility of tβh females is rescued by restored Tβh expression in a subset of neurons including the TAG neurons that innervate the reproductive system (Monastirioti, 1995). Consistently, octopamine, when applied to the dissected reproductive system, modulates muscle activities in a tissue-specific manner: it enhances muscle contraction in the ovary but inhibits it in the oviduct (Rodriguez-Valentin, 2006; Middleton, 2006). This suggests that distinct octopamine receptors present in the ovary and oviduct mediate the opposite actions of octopamine on muscle activity (Lee, 2009).

Drosophila has four known octopamine receptors: OAMB, Octβ1R, Octβ2R and Octβ3R (Han, 1998; Maqueira, 2005; Balfanz, 2005). The oamb gene encodes two isoforms OAMB-K3 (K3) and OAMB-AS (AS), which are produced by alternative splicing of the last exon, and differ in the third cytoplasmic loop and downstream sequence. Both K3 and AS transcripts are found in the brain, TAG and reproductive system (Lee, 2003; Han, 1998). When assayed in the heterologous cell lines, both isoforms activate an increase in intracellular Ca2+ (Han, 1998; Balfanz, 2005) while K3 also stimulates a cAMP increase (Han 1998). This implies that the two isoforms may activate distinct combinations of signal transduction pathways in vivo. To investigate OAMB's in vivo functions, several oamb mutants defective in both K3 and AS have been generated, and their prominent phenotype is female sterility (Lee, 2003). While oamb mutant females show normal mating, they are impaired in ovulation, causing abnormal retention of mature eggs in the ovary (Lee, 2003). This raises several important questions regarding mechanism of OAMB activity: where (brain, TAG or reproductive system) does OAMB regulate ovulation? Which isoform is critical for this process and what are the downstream signals? This study shows that the critical site for the OAMB's function in ovulation is the oviduct epithelium, in which transgenic expression of either K3 or AS isoform is sufficient to rescue the oamb female's ovulation defect. Moreover, OAMB recruits CaMKII as a key downstream effector for this function (Lee, 2009).

Octopamine, as a major neurotransmitter, neuromodulator and neurohormone, regulates diverse physiological processes in invertebrates that include sensory information processing, egg-laying, fight or flight responses, and complex neural functions such as learning and memory (Roeder, 2005). These astonishingly diverse effects of octopamine are initiated by the binding of octopamine to G-protein-coupled receptors expressed in distinct tissue or cell types; however, very little is known about relevant octopamine receptors and underlying cellular mechanisms that mediate octopamine's physiological functions. This work has shown that OAMB regulates ovulation in the oviduct epithelium and recruits CaMKII for this function. This role of OAMB is physiological, as opposed to developmental, since restored OAMB expression in the oviduct epithelium at the adult stage is sufficient for reinstating ovulation in oamb females. This is consistent with the findings observed in the octopamine-less dTdc2 and tβh females, in which feeding octopamine only at the adult stage rescues the sterility phenotype of both mutants (Cole, 2005; Lee, 2009 and references therein).

Sex peptides transferred to the female during copulation enhance egg-laying upon binding to the receptor SPR expressed in the Fruitless neurons. The mechanism by which the Fruitless neurons stimulate egg-laying is unknown; however, octopaminergic neurons in the TAG likely represent a downstream target since the egg-laying phenotype of tβh females is rescued by restored TβH expression in these neurons (Monastirioti, 2003). The TAG octopaminergic neurons project axons to various areas in the reproductive track including the ovary, lateral and common oviducts, sperm storage organs and the uterus (Rodriguez-Valentin, 2006; Middleton, 2003). Mating induces distinctive changes in vesicle release at the nerve terminals in different areas of the reproductive track (Heifetz, 2004), some of which may represent the TAG octopamine neuronal activities. In the dissected reproductive system, octopamine application augments the amplitude of myogenic contractions of the peritoneal sheath in the ovary while it inhibits stimulated muscle contractions of the oviduct (Rodriguez-Valentin, 2006; Middleton, 2003). These opposite effects of octopamine may be crucial for coordinated constriction and relaxation of the ovary and oviduct, respectively, in transferring a mature egg to the uterus. OAMB may serve as a receptor processing the octopamine's input in the oviduct while another octopamine receptor may mediate the constriction signal in the ovarian peritoneal sheath, which lacks OAMB expression (Lee, 2009).

Remarkably, OAMB's activity is required in the epithelium rather than the muscle for normal ovulation. Consistent with this, the histochemical analysis reported here reveals extensive innervation of the TAG octopamine neuronal processes into the oviduct epithelial layer where both OAMB isoforms are enriched, in addition to the muscle. This raises an important question regarding the nature of an OAMB's role in ovulation. While no information is available on the oviduct epithelium in Drosophila or other insects, studies of the mammalian oviduct indicate active roles of the epithelium in fluid secretion and ciliary activity for gamete and embryo transport. Similarly, it is possible that OAMB in the Drosophila oviduct epithelium is involved in regulating fluid secretion to establish proper luminal environment and possibly ciliary action for egg transport. The capacity of either OAMB-K3 or OAMB-AS to reinstate ovulation in oamb females strongly implicates intracellular Ca²⁺ rather than cAMP as a downstream effector. This is corroborated by findings demonstrating CaMKII as a key epithelial component downstream of OAMB. It is uncertain whether individual isoforms or two isoforms together have comparable efficacies in activating CaMKII and ovulation. Future studies employing quantitative manipulation of transgenic OAMB expression may clarify this issue. Taken together, the epithelial OAMB stimulated upon mating likely activates CaMKII via increased intracellular Ca²⁺, which may in turn trigger biochemical changes necessary for fluid secretion. Potential molecules involved in this process may include transporters, ion channels, Na⁺-K⁺-ATPase and the molecules involved in cilia movements. In the absence of OAMB, epithelial cell activities and fluid may be inadequate for egg movement, leading to ovulation failure. Since octopamine induces relaxation in the dissected oviduct, relaxation may involve another octopamine receptor in the muscle, and concerted activities of OAMB and a muscle receptor may be crucial for successful egg transport. This working model is currently under test (Lee, 2009).

Octopamine regulates oviduct activities in other insects as well. In the locust oviduct, octopamine inhibits the basal tonus and neurally evoked muscle contractions, which are mediated by cAMP-dependent mechanisms (Nykamp, 2000; Orchard, 1986). These effects of octopamine may be mediated by an OAMB-like receptor with the different intracellular effector cAMP. Alternatively, they may involve another octopamine receptor(s) present in the muscle. Drosophila has three octopamine receptors (OctβR1, 2 and 3) that can also stimulate cAMP increases (Evans, 2005). Spatial expression patterns of three OctβRs are as yet unknown. It is conceivable that an OctβR or OctβR-like receptor, possibly present in the Drosophila or locust oviduct muscle, respectively, is additionally involved in ovulation by inducing muscle relaxation through a cAMP signaling pathway. At present, molecular components and cellular pathways controlling ovulation are largely unknown and likewise very little is known about the oviduct functions and mechanisms. The current findings uncover the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. Future studies to identify additional downstream effectors of OAMB and their functions should help further understanding of the important reproductive process ovulation and provide novel insights into the development of effective insecticides. Typically, intracellular signals activated by G-protein-coupled receptors are characterized in in vitro cell lines. This study has identified the intracellular signal activated by the G-protein-coupled receptor OAMB in vivo that has functional significance. Similar approaches could be applied to other receptors to investigate rather poorly defined cellular mechanisms that G-protein-coupled receptors activate for their in vivo functions (Lee, 2009).

Norepinephrine, a mammalian counterpart of octopamine, also plays profound roles in female reproduction by acting on the reproductive and nervous systems. Sympathetic nerve terminals containing norepinephrine innervate the ovaries, oviducts, and uterus. Moreover, norepinephrine levels in the human fallopian tube vary in a region- and estrous cycle-dependent manner being the highest in the isthmus and the fimbriated end at the time of ovulation. When assayed in vitro, adrenergic receptor agonists not only modulate oviduct muscle activities but they also stimulate fluid secretion possibly via Ca²⁺-dependent mechanisms. Oviduct fluid in mammals is critical for egg transport, maturation and fertilization; however, the cellular process regulating its secretion is largely unknown. Damage in the oviduct epithelium is associated with pelvic inflammatory disorder, leading to hydrosalpinx formation and reduced fertility. Thus, enhanced understanding of physiological and cellular factors and processes controlling oviduct fluid will provide significant insights into healthy reproduction as well as impaired fertility associated with pelvic inflammatory disorder and other related disorders (Lee, 2009).

Octopamine receptor OAMB is required for ovulation in Drosophila melanogaster

Octopamine is a major monoamine in invertebrates and affects many physiological processes ranging from energy metabolism to complex behaviors. Octopamine binds to receptors located on various cell types and activates distinct signal transduction pathways to produce these diverse effects. One of the Drosophila octopamine receptors named OAMB produces increases in cAMP and intracellular Ca2+ upon ligand binding. It is expressed at high levels in the brain. To explore OAMB's physiological roles, deletions were generated in the OAMB locus. The resultant oamb mutants were viable without gross anatomical defects. The oamb females displayed normal courtship and copulation; however, they were impaired in ovulation with many mature eggs retained in their ovaries. RT-PCR, in situ hybridization, and expression of a reporter gene revealed that OAMB was also expressed in the thoracicoabdominal ganglion, the female reproductive system, and mature eggs in the ovary. Moreover, analysis of various alleles pinpointed the requirement for OAMB in the body, but not in the brain, for female fecundity. The novel expression pattern of OAMB and its genetic resource described in this study will help advance understanding on how the neuromodulatory or endocrine system controls reproductive physiology and behavior (Lee, 2003; Full text of article).

A novel octopamine receptor with preferential expression in Drosophila mushroom bodies

Octopamine is a neuromodulator that mediates diverse physiological processes in invertebrates. In some insects, such as honeybees and fruit flies, octopamine has been shown to be a major stimulator of adenylyl cyclase and to function in associative learning. To identify an octopamine receptor mediating this function in Drosophila, putative biogenic amine receptors were cloned by a novel procedure using PCR and single-strand conformation polymorphism. One new receptor, octopamine receptor in mushroom bodies (OAMB), was identified as an octopamine receptor because human and Drosophila cell lines expressing OAMB showed increased cAMP and intracellular Ca2+ levels after octopamine application. Immunohistochemical analysis using an antibody made to the receptor revealed highly enriched expression in the mushroom body neuropil and the ellipsoid body of central complex, brain areas known to be crucial for olfactory learning and motor control, respectively. The preferential expression of OAMB in mushroom bodies and its capacity to produce cAMP accumulation suggest an important role in synaptic modulation underlying behavioral plasticity (Han, 1998. Full text of article).

Search PubMed for articles about Drosophila Oamb

Balfanz, S., Strunker, T., Frings, S. and Baumann, A. (2005). A family of octopamine receptors that specifically induce cyclic AMP production or Ca2+ release in Drosophila melanogaster. J. Neurochem. 93: 440-451. PubMed Citation: 15816867

Cole, S. H., et al. (2005). Two functional but noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility. J. Biol. Chem. 280: 14948-14955. PubMed Citation: 15691831

Evans, P. D. and Maqueira, B. (2005). Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors. Invert. Neurosci. 5: 111-118. PubMed Citation: 16211376

Han, K. A., Millar, N. S. and Davis, R. L. (1998). A novel octopamine receptor with preferential expression in Drosophila mushroom bodies. J. Neurosci. 18: 3650-3658. PubMed Citation: 9570796

Heifetz, Y. and Wolfner, M. F. (2004). Mating, seminal fluid components, and sperm cause changes in vesicle release in the Drosophila female reproductive tract. Proc. Natl. Acad. Sci. 101: 6261-6266. PubMed Citation: 15071179

Herndon, L. A. and Wolfner, M. F. (1995). A Drosophila seminal fluid protein, Acp26Aa, stimulates egg laying in females for 1 day after mating. Proc. Natl. Acad. Sci. 92: 10114-10118. PubMed Citation: 7479736

Lee, H. G., Seong, C. S., Kim, Y. C., Davis, R. L. and Han, K. A. (2003). Octopamine receptor OAMB is required for ovulation in Drosophila melanogaster. Dev. Biol. 264: 179-190. PubMed Citation: 14623240

Lee, H. G., Rohila, S. and Han, K. A. (2009). The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium. PLoS One 4(3): e4716. PubMed Citation: 19262750

Maqueira, B., Chatwin, H. and Evans, P. D. (2005). Identification and characterization of a novel family of Drosophila beta-adrenergic-like octopamine G-protein coupled receptors. J. Neurochem. 94: 547-560. PubMed Citation: 15998303

Middleton, C. A., et al. (2006). Neuromuscular organization and aminergic modulation of contractions in the Drosophila ovary. BMC Biol. 4: 17. PubMed Citation: 16768790

Monastirioti, M., et al. (1995). Octopamine immunoreactivity in the fruit fly Drosophila melanogaster. J. Comp. Neurol. 356: 275-287. PubMed Citation: 7629319

Monastirioti, M. (2003). Distinct octopamine cell population residing in the CNS abdominal ganglion controls ovulation in Drosophila melanogaster. Dev. Biol. 264: 38-49. PubMed Citation: 14623230

Nykamp, D. A. and Lange, A. B. (2000). Interaction between octopamine and protolin on the oviducts of Locusta migratoria. J. Insect Physiol. 46: 809-816. PubMed Citation: 10742530

Orchard, I. and Lange, A. B. (1986). Neuromuscular transmission in an insect visceral muscle. J. Neurobiol. 17: 359-372. PubMed Citation: 2877049

Rodriguez-Valentin, R., et al. (2006). Oviduct contraction in Drosophila is modulated by a neural network that is both, octopaminergic and glutamatergic. J. Cell/ Physiol. 209: 183-198. PubMed Citation: 16826564

Roeder, T. (2005). Tyramine and octopamine: ruling behavior and metabolism. Annu. Rev. Entomol. 50: 447-477. PubMed Citation: 15355245

Sinakevitch I, Strausfeld NJ. Comparison of octopamine-like immunoreactivity in the brains of the fruit fly and blow fly. J. Comp. Neurol. 494: 460-475. PubMed Citation: 16320256

date revised: 30 December 2009

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