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.

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.

Pigment-dispersing factor (PDF) is a neuropeptide that plays a prominent role in the circadian clock of several insects. The cockroach Leucophaea maderae was the first animal where the site of a biological clock could be located, and still is a focal point of circadian research. Although detailed studies on the action of pigment-dispersing factor and the distribution of PDF-like immunoreactivity in the L. maderae brain exist, a native pigment-dispersing factor of this species has not been characterized so far. The authentic Lem-PDF was isolated from L. maderae by a combination of high performance liquid chromatography, crab pigment-dispersion bioassay and an immunosorbent assay. Mass spectrometric characterization and the conserved sequence of pigment-dispersing factor in orthopteromorphan insects suggest that Lem-PDF has the sequence NSEXINSLLGLPKVLNDAa (where X= I or L). Lem-PDF is thus identical to either Periplaneta americana PDF or Acheta domesticus PDF. Detailed analysis of PDF-like immunofluorescence in different regions of the brain suggests that there are no drastic daily changes in the amount of pigment-dispersing factor as occur in Drosophila melanogaster, which might be explained by a lack of circadian pigment-dispersing factor release and production, or by phase differences between the pigment-dispersing factor neurons.