Circadian clocks play vital roles in the control of daily rhythms in physiology and behavior of animals. In Drosophila, analysis of the molecular and behavioral rhythm has shown that the master clock neurons are entrained by sensory inputs and are synchronized with other clock neurons. However, little is known about the neuronal circuits of the Drosophila circadian system and the neurotransmitters that act on the clock neurons. Here, we provide evidence for a new neuronal input pathway to the master clock neurons, s-LN(v)s, in Drosophila that utilizes GABA as a slow inhibitory neurotransmitter. We monitored intracellular calcium levels in dissociated larval s-LN(v)s with the calcium-sensitive dye Fura-2. GABA decreased intracellular calcium in the s-LN(v)s and blocked spontaneous oscillations in calcium levels. The duration of this response was dose-dependent between 1 nM and 100 microM. The response to GABA was blocked by a metabotropic GABA(B) receptor (GABA(B)-R) antagonist, CGP54626, but not by an ionotropic receptor antagonist, picrotoxin. The GABA(B)-R agonist, 3-APMPA, produced a response similar to GABA. An antiserum against one of the Drosophila GABA(B)-Rs (GABA(B)-R2) labeled the dendritic regions of the s-LN(v)s in both adults and larvae, as well as the dissociated s-LN(v)s. We found that some GABAergic processes terminate at the dendrites of the LN(v)s, as revealed by GABA immunostaining and a GABA-specific GAL4 line (GAD1-gal4). Our results suggest that the s-LN(v)s receive slow inhibitory GABAergic inputs that decrease intracellular calcium of these clock neurons and block their calcium cycling. This response is mediated by postsynaptic GABA(B) receptors.

Identification of the neurotransmitters in clock neurons is critical for understanding the circuitry of the neuronal network that controls the daily behavioral rhythms in Drosophila. Except for the neuropeptide pigment-dispersing factor, no neurotransmitters have been clearly identified in the Drosophila clock neurons. Here we show that glutamate and its metabotropic receptor, DmGluRA, are components of the clock circuitry and modulate the rhythmic behavior pattern of Drosophila. The dorsal clock neurons, DN1s in the larval brain and some DN1s and DN3s in the adult brain, were immunolabeled with antibodies against Drosophila vesicular glutamate transporter (DvGluT), suggesting that they are glutamatergic. Because the DN1s may communicate with the primary pacemaker neurons, s-LN(v)s, we tested glutamate responses of dissociated larval s-LN(v)s by means of calcium imaging. Application of glutamate dose dependently decreased intracellular calcium in the s-LN(v)s. Pharmacology of the response suggests the presence of DmGluRA on the s-LN(v)s. Antibodies against DmGluRA labeled dissociated s-LN(v)s and the LN(v) dendrites in the intact larval and adult brain. The role of metabotropic glutamate signaling was tested in behavior assays in transgenic larvae and flies with altered DmGluRA expression in the LN(v)s and other clock neurons. Larval photophobic behavior was enhanced in DmGluRA mutants. For adults, we could induce altered activity patterns in the dark phase under LD conditions and increase the period during constant darkness by knockdown of DmGluRA expression in LN(v)s. Our results suggest that a glutamate signal from some of the DNs modulates the rhythmic behavior pattern via DmGluRA on the LN(v)s in Drosophila.

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in insects and is widely distributed in the central nervous system (CNS). GABA acts on ion channel receptors (GABA(A)R) for fast inhibitory transmission and on G-protein-coupled ones (GABA(B)R) for slow and modulatory action. We used immunocytochemistry to map GABA(B)R sites in the Drosophila CNS and compared the distribution with that of the GABA(A)R subunit RDL. To identify GABAergic synapses, we raised an antiserum to the vesicular GABA transporter (vGAT). For general GABA distribution, we utilized an antiserum to glutamic acid decarboxylase (GAD1) and a gad1-GAL4 to drive green fluorescent protein. GABA(B)R-immunoreactive (IR) punctates were seen in specific patterns in all major neuropils of the brain. Most abundant labeling was seen in the mushroom body calyces, ellipsoid body, optic lobe neuropils, and antennal lobes. The RDL distribution is very similar to that of GABA(B)R-IR punctates. However, the mushroom body lobes displayed RDL-IR but not GABA(B)R-IR material, and there were subtle differences in other areas. The vGAT antiserum labeled punctates in the same areas as the GABA(B)R and appeared to display presynaptic sites of GABAergic neurons. Various GAL4 drivers were used to analyze the relation between GABA(B)R distribution and identified neurons in adults and larvae. Our findings suggest that slow GABA transmission is very widespread in the Drosophila CNS and that fast RDL-mediated transmission generally occurs at the same sites.

Light entrains the biological clock both in adult and larval Drosophila melanogaster. The Bolwig organ photoreceptors most likely constitute one substrate for this light entrainment in larvae. Acetylcholine (ACh) has been suggested as the neurotransmitter in these photoreceptors, but there is no evidence that ACh signaling is involved in photic input onto circadian pacemaker neurons. Here we demonstrate that the putative targets of the Bolwig photoreceptors, the PDF-containing clock neurons (LNs), in the larval brain express functional ACh receptors (AChRs). With the use of GAL4-UAS-driven expression of green fluorescent protein (GFP), we were able to identify LNs in dissociated cell culture. After loading with the Ca(2+)-sensitive dye fura-2, we monitored changes in intracellular Ca(2+) levels ([Ca(2+)](i)) in GFP-marked LNs while applying candidate neurotransmitters. ACh induced transient increases in [Ca(2+)](i) at physiological concentrations. These increases were dependent on extracellular Ca(2+) and Na(+) and were likely caused by activation of voltage-dependent Ca(2+) channels. Application of nicotinic and muscarinic agonists and antagonists showed that the AChRs on cultured LNs have a nicotinic pharmacology. Antibodies to several subunits of nicotinic AChRs (nAChRs) labeled the putative contact site of the Bolwig organ axon terminals with the dendrites of LNs, as well as dissociated LNs in culture. Our findings support a role of ACh as input factor onto the LNs and suggest that Ca(2+) is used as a second messenger mediating cholinergic input within the LNs. Experiments using a more general GAL4-UAS-driven expression of GFP showed that functional expression of nAChRs is a widespread phenomenon in peptidergic neurons.

Several sets of clock neurons cooperate to generate circadian activity rhythms in Drosophila melanogaster. To extend the knowledge on neurotransmitters in the clock circuitry, we analyzed the distribution of some biogenic amines in relation to identified clock neurons. This was accomplished by employing clock neuron-specific GAL4 lines driving green fluorescent protein (GFP) expression, combined with immunocytochemistry with antisera against serotonin, histamine, and tyrosine hydroxylase (for dopamine). In the larval and adult brain, serotonin-immunoreactive (-IR) neuron processes are in close proximity of both the dendrites and the dorsal terminals of the major clock neurons, the s-LN(v)s. Additionally, the terminals of the l-LN(v) clock neurons and serotonergic processes converge in the distal medulla. No histamine (HA)-IR processes contact the s-LN(v)s in the larval brain, but possibly impinge on the dorsal clock neurons, DN2. In the adult brain, HA-IR axons of the extraocular eyelet photoreceptors terminate on the dendritic branches of the LN(v)s. A few tyrosine hydroxylase (TH)-IR processes were seen close to the dorsal terminals of the s-LN(v)s, but not their dendrites, in the larval and adult brain. TH-IR processes also converge with the distal medulla branches of the l-LN(v)s in adults. None of the monoamines was detectable in the different clock neurons. By using an imaging system to monitor intracellular Ca(2+) levels in dissociated GFP-labeled larval s-LN(v)s, loaded with Fura-2, we demonstrated that application of serotonin induced dose-dependent decreases in Ca(2+). Thus, serotonergic neurons form functional inputs on the s-LN(v)s in the larval brain and possibly also in adults.