BRP-170 and BRP190 isoforms of Bruchpilot protein differentially contribute to the frequency of synapses and synaptic circadian plasticity in the visual system of Drosophila.

In the first optic neuropil (lamina) of the optic lobe of Drosophila melanogaster, two classes of synapses, tetrad and feedback, show daily rhythms in the number and size of presynaptic profiles examined at the level of transmission electron microscopy (TEM). Number of tetrad presynaptic profiles increases twice a day, once in the morning and again in the evening, and their presynaptic ribbons are largest in the evening. In contrast, feedback synapses peak at night. The frequency of synapses is correlated with size of the presynaptic element measured as the platform size of so-called T-bars, with T-bar platforms being largest with increasing synapse frequency. The large scaffold protein Bruchpilot (BRP) is a major essential constituent of T-bars, with two major isoforms of 190 and 170 kD forming T-bars of the peripheral neuromuscular junctions (NMJ) synapses and in the brain. In addition to the analysis of cyclic plasticity of tetrad and feedback synapses in wild-type flies, we used TEM to examine daily changes in the size and distribution of synapses within isoform-specific BRP mutants, expressing BRP-190 (BRPΔ170) or BRP-170 (BRPΔ190) only. We found that the number and circadian plasticity of synapses depends on both isoforms. In the BRPΔ190 lacking BRP-190 there was almost 50% less tetrad synapses demonstrable than when both isoforms were present. The lack of BRP-170 and BRP-190 increased and decreased, respectively the number of feedback synapses, indicating that BRP-190 forms most of the feedback synapses. In both mutants, the daily plasticity of tetrad and feedback presynaptic profiles was abolished, except for feedback synapses in BRPΔ190. The oscillations in the number and size of presynaptic elements seem to depend on a different contribution of BRP isoforms in a presynaptic element at different time during the day and night and at various synapse types. The participation of both BRP isoforms may vary in different classes of synapses.

External and circadian inputs modulate synaptic protein expression in the visual system of Drosophila melanogaster.

In the visual system of Drosophila melanogaster the retina photoreceptors form tetrad synapses with the first order interneurons, amacrine cells and glial cells in the first optic neuropil (lamina), in order to transmit photic and visual information to the brain. Using the specific antibodies against synaptic proteins; Bruchpilot (BRP), Synapsin (SYN), and Disc Large (DLG), the synapses in the distal lamina were specifically labeled. Then their abundance was measured as immunofluorescence intensity in flies held in light/dark (LD 12:12), constant darkness (DD), and after locomotor and light stimulation. Moreover, the levels of proteins (SYN and DLG), and mRNAs of the brp, syn, and dlg genes, were measured in the fly's head and brain, respectively. In the head we did not detect SYN and DLG oscillations. We found, however, that in the lamina, DLG oscillates in LD 12:12 and DD but SYN cycles only in DD. The abundance of all synaptic proteins was also changed in the lamina after locomotor and light stimulation. One hour locomotor stimulations at different time points in LD 12:12 affected the pattern of the daily rhythm of synaptic proteins. In turn, light stimulations in DD increased the level of all proteins studied. In the case of SYN, however, this effect was observed only after a short light pulse (15 min). In contrast to proteins studied in the lamina, the mRNA of brp, syn, and dlg genes in the brain was not cycling in LD 12:12 and DD, except the mRNA of dlg in LD 12:12. Our earlier results and obtained in the present study showed that the abundance of BRP, SYN and DLG in the distal lamina, at the tetrad synapses, is regulated by light and a circadian clock while locomotor stimulation affects their daily pattern of expression. The observed changes in the level of synaptic markers reflect the circadian plasticity of tetrad synapses regulated by the circadian clock and external inputs, both specific and unspecific for the visual system.

Circadian expression of the presynaptic active zone protein Bruchpilot in the lamina of Drosophila melanogaster.

In the fly's visual system, the morphology of cells and the number of synapses change during the day. In the present study we show that in the first optic neuropil (lamina) of Drosophila melanogaster, a presynaptic active zone protein Bruchpilot (BRP) exhibits a circadian rhythm in abundance. In day/night (or light/dark, LD) conditions the level of BRP increases two times, in the morning and in the evening. The same pattern of changes in the BRP level was detected in whole brain homogenates, thus indicating that the majority of synapses in the brain peaks twice during the day. However, these two peaks in BRP abundance, measured as the fluorescence intensity of immunolabeling, seem to be regulated differently. The peak in the morning is predominantly regulated by light and involves the transduction pathway in the retina photoreceptors. This peak is present neither in wild-type Canton-S flies in constant darkness (DD), nor in norpA(7) phototransduction mutant in LD. However, it also depends on the clock gene per, because it is abolished in the per(0) arrhythmic mutant. In turn, the peak of BRP in the evening is endogenously regulated by an input from the pacemaker located in the brain. This peak is present in Canton-S flies in DD, as well as in the norpA(7) mutant in LD, but is absent in per(01), tim,(01) and cry(01) mutants in LD. In addition both peaks seem to depend on clock gene-expressing photoreceptors and glial cells of the visual system.

Cyclical expression of Na+/K+-ATPase in the visual system of Drosophila melanogaster.

In the first (lamina) and second (medulla) optic neuropils of Drosophila melanogaster, sodium pump subunit expression changes during the day and night, controlled by a circadian clock. We examined alpha-subunit expression from the intensity of immunolabeling. For the beta-subunit, encoded by Nervana 2 (Nrv2), we used Nrv2-GAL4 to drive expression of GFP, and measured the resultant fluorescence in whole heads and specific optic lobe cells. All optic neuropils express the alpha-subunit, highest at the beginning of night in both lamina and medulla in day/night condition and the oscillation was maintained in constant darkness. This rhythm was lacking in the clock arrhythmic per(0) mutant. GFP driven by Nrv2 was mostly detected in glial cells, mainly in the medulla. There, GFP expression occurs in medulla neuropil glia (MNGl), which express the clock gene per, and which closely contact the terminals of clock neurons immunoreactive to pigment dispersing factor. GFP fluorescence exhibited circadian oscillation in whole heads from Nrv2-GAL4+UAS-S65T-GFP flies, although significant GFP oscillations were lacking in MNGl, as they were for both subunit mRNAs in whole-head homogenates. In the dissected brain tissues, however, the mRNA of the alpha-subunit showed a robust daily rhythm in concentration changes while changes in the beta-subunit mRNA were weaker and not statistically significant. Thus in the brain, the genes for the sodium pump subunits, at least the one encoding the alpha-subunit, seem to be clock-controlled and the abundance of their corresponding proteins mirrors daily changes in mRNA, showing cyclical accumulation in cells.