ABSTRACT
Store operated calcium entry (SOCE) is thought to primarily regulate calcium homeostasis in neurons. Subsequent to identification of Orai as the SOCE channel in nonexcitable cells, investigation of Orai function in neurons demonstrated a requirement for SOCE in Drosophila flight. Here, by analysis of an Orai mutant and by controlled expression of a dominant-negative Drosophila Orai transgene, we show that Orai-mediated SOCE is required in dopaminergic interneurons of the flight circuit during pupal development. Expression of dominant-negative Orai in dopaminergic neurons of pupae abolished flight. The loss of Orai-mediated SOCE alters transcriptional regulation of dopaminergic neurons, leading to downregulation of the enzyme tyrosine hydroxylase, which is essential for dopamine synthesis, and the dopamine transporter, which is required for dopamine uptake after synaptic release. These studies suggest that modulation of SOCE could serve as a novel mechanism for restoring dopamine levels in dopaminergic neurons. SIGNIFICANCE STATEMENT: The specificity of an animal's response to an environmental stimulus is determined in part by the release of neurotransmitters, which are sensed by responding neurons through cognate receptors on their surface. One way by which neurons respond is through release of calcium from intracellular stores followed by store refilling from extracellular calcium sources. This mechanism is called store-operated calcium entry (SOCE). The function of SOCE in neurons has been debated. Here we describe a new function for SOCE in the regulation of neurotransmitter levels in Drosophila flight neurons. This cell-signaling mechanism is required to maintain optimal levels of a key enzyme for dopamine synthesis and may serve as a mechanism for restoring dopamine levels in relevant pathological conditions.
Subject(s)
Calcium/metabolism , Drosophila Proteins/metabolism , Flight, Animal/physiology , Gene Expression Regulation, Developmental/genetics , Membrane Proteins/metabolism , Mutation/genetics , Acetyl-CoA Carboxylase/genetics , Acetyl-CoA Carboxylase/metabolism , Animals , Animals, Genetically Modified , Calcium Signaling/genetics , Cells, Cultured , Dopamine Plasma Membrane Transport Proteins/metabolism , Dopaminergic Neurons/metabolism , Drosophila , Drosophila Proteins/genetics , Flow Cytometry , Larva , Membrane Proteins/genetics , Neural Pathways/physiology , ORAI1 Protein , Pupa , Rats , Tyrosine 3-Monooxygenase/metabolismABSTRACT
The final identity of a differentiated neuron is determined by multiple signaling events, including activity dependent calcium transients. Non-canonical Frizzled2 (Fz2) signaling generates calcium transients that determine neuronal polarity, neuronal migration, and synapse assembly in the developing vertebrate brain. Here, we demonstrate a requirement for Fz2/Ca(2+) signaling in determining the final differentiated state of a set of central brain dopaminergic neurons in Drosophila, referred to as the protocerebral anterior medial (PAM) cluster. Knockdown or inhibition of Fz2/Ca(2+) signaling during maturation of the flight circuit in pupae reduces Tyrosine Hydroxylase (TH) expression in the PAM neurons and affects maintenance of flight. Thus, we demonstrate that Fz2/Ca(2+) transients during development serve as a pre-requisite for normal adult behavior. Our results support a neural mechanism where PAM neuron send projections to the α' and ß' lobes of a higher brain centre, the mushroom body, and function in dopaminergic re-inforcement of flight.
Subject(s)
Calcium Signaling , Calcium/metabolism , Drosophila Proteins/genetics , Frizzled Receptors/genetics , Animals , Brain/growth & development , Brain/metabolism , Cell Movement , Cell Polarity , Dopaminergic Neurons/cytology , Dopaminergic Neurons/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Flight, Animal/physiology , Frizzled Receptors/metabolism , Gene Expression Regulation, Developmental , Genetic Complementation Test , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mushroom Bodies/growth & development , Mushroom Bodies/metabolism , Pupa/genetics , Pupa/growth & development , Pupa/metabolism , Stromal Interaction Molecule 1 , Synapses/physiology , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Insect flight is regulated by various sensory inputs and neuromodulatory circuits which function in synchrony to control and fine-tune the final behavioral outcome. The cellular and molecular bases of flight neuromodulatory circuits are not well defined. In Drosophila melanogaster, it is known that neuronal IP3 receptor mediated Ca²âº signaling and store-operated Ca²âº entry (SOCE) are required for air-puff stimulated adult flight. However, G-protein coupled receptors (GPCRs) that activate intracellular Ca²âº signaling in the context of flight are unknown in Drosophila. We performed a genetic RNAi screen to identify GPCRs that regulate flight by activating the IPIP3 receptor. Among the 108 GPCRs screened, we discovered 5 IPIP3/Ca²âº linked GPCRs that are necessary for maintenance of air-puff stimulated flight. Analysis of their temporal requirement established that while some GPCRs are required only during flight circuit development, others are required both in pupal development as well as during adult flight. Interestingly, our study identified the Pigment Dispersing Factor Receptor (PdfR) as a regulator of flight circuit development and as a modulator of acute flight. From the analysis of PdfR expressing neurons relevant for flight and its well-defined roles in other behavioral paradigms, we propose that PdfR signaling functions systemically to integrate multiple sensory inputs and modulate downstream motor behavior.
