Circadian pacemaker neurons change synaptic contacts across the day

Abstract

Daily cycles of rest and activity are a common example of circadian control of physiology. In Drosophila, rhythmic locomotor cycles rely on the activity of 150-200 neurons grouped in seven clusters [1, 2]. Work from many laboratories points to the small ventral lateral neurons (sLNvs) as essential for circadian control of locomotor rhythmicity [3-7]. sLNv neurons undergo circadian remodeling of their axonal projections, opening the possibility for a circadian control of connectivity of these relevant circadian pacemakers [8]. Here we show that circadian plasticity of the sLNv axonal projections has further implications than mere structural changes. First, we found that the degree of daily structural plasticity exceeds that originally described [8], underscoring that changes in the degree of fasciculation as well as extension or pruning of axonal terminals could be involved. Interestingly, the quantity of active zones changes along the day, lending support to the attractive hypothesis that new synapses are formed while others are dismantled between late night and the following morning. More remarkably, taking full advantage of the GFP reconstitution across synaptic partners (GRASP) technique [9], we showed that, in addition to new synapses being added or removed, sLNv neurons contact different synaptic partners at different times along the day. These results lead us to propose that the circadian network, and in particular the sLNv neurons, orchestrates some of the physiological and behavioral differences between day and night by changing the path through which information travels.

Figure 1. Severe morphological and synaptic changes occur during the dark to light transition

(A)Representative confocal images taken at CT2, CT14 and CT22. During early subjective day (CT2) axonal projections are more complex and extended, reaching further towards the medial region, while at CT22 PDF projections are less complex (as in CT14) and appear shorter. (B-C) Quantitation of total axonal crosses (B) and the longest axonal branch (C) at CT2, CT6, CT14, CT18 and CT22 of control brains (pdf-GS>mCD8GFP). Dissections were performed on the 4^th day of constant darkness (DD4). Dark gray represents subjective night while light grey, subjective day. * indicates significant differences with p<0.05. Statistical analysis included Blocked ANOVA (Total axonal crosses p=0.0002; circuit length p=0.0417) with Tukey post-hoc test (p<0.05, (Total axonal crosses least significant difference = 3.40; circuit length least significant difference = 10.98 μm). (D) Representative confocal images of dorsal sLNvs projections from cultured brains. Brains were cultured 72 h and imaged 24 h post dissection (PD, left panel), which equals CT14, and 36 h PD (CT2, right panel). A fasciculation/defasciculation process could be appreciated in the principal branches (arrows), while in secondary neurites different phenomena were observed: addition/retraction (asterisk) and positional changes (arrowhead). (E) Quantitation of changes seen in different cultured brains (n=6). (F) Representative confocal images of fly brains stained for BRP-RFP (white) and PDF (magenta) dissected at CT2, CT14 and CT22 on DD4. (G-H) Quantitation of BRP⁺ active zones (G) and the total area covered by them (H). Control pdf-GS>brp^RFP flies display circadian changes in BRP⁺ active zones as well as the area covered by BRP⁺ immunoreactivity. Significant differences were found in both variables between subjective day and night, but not between timepoints taken at nighttime. Same letters indicate no significant differences. Statistical analysis included one way ANOVA (BRP⁺ active zones p=0.0069; BRP⁺ area p<0.0001) with Tukey post-hoc test (p<0.05, BRP⁺ active zones least significant difference = 6.99; BRP⁺ area least significant difference = 3.35 μm²). In all cases the scale bar represents 10 μm.

Figure 2. GRASP analysis on putative clock partners reveals constant and plastic changes in sLNv connectivity

Images represent examples of putative synaptic partners of PDF neurons. Expression profiles of (A) dClock4.1-Gal4 to light up DN1p neurons and (B) Mai179Gal4;pdfG80 expression on a restricted subset of circadian-relevant neurons including the 5^th sLNv, up to 4 LNds (and PI cells). PDF and GFP signals are shown in magenta and green, respectively. (A1-A3) Representative confocal images of a pdf-lexA>lexAop-CD4::GFP¹¹/dClock4.1-Gal4>UAS-CD4GFP¹¹⁰ brain dissected during early day (ZT2, A1), early night (ZT14, A2) and late night (ZT22, A3). (Al′-A3′) Reconstituted GFP⁺ signal is shown; the structure of PDF projections is outlined by a dashed line (encircling the PDF signal) to improve visualization of the reconstituted GFP. GFP⁺ signal was observed at all timepoints analyzed. (B1-B3 and Bl′-B3′) Intersection between PDF and Mai179Gal4;pdfG80 neurons (the so-called evening oscillator, [8]). The reconstituted signal changes across the day, becoming more pervasive at nighttime. Scale bar represents 10 μm unless otherwise noted. (C) Quantitative analysis confirms constant contacts between sLNvs and DN1p clusters, but plastic ones between sLNvs and the evening oscillator, with a statistically significant increase at ZT22 (Kruskal-Wallis test p<0.01).

Figure 3. A GRASP screen uncovers changes in connectivity to non-circadian targets

Images represent examples of putative synaptic partners of PDF neurons contacting them in different time-windows; throughout the day (A), during ZT2 (B) or during ZT14 (C). (A-C). Expression profiles of 11-8 (A), 3-86 (B), 4-59 (C), and OK¹⁰⁷ (D) neuronal clusters. PDF and GFP signals are shown in magenta and green, respectively. 3-86 is expressed in the PI and sends neurites proximal to sLNvs dorsal projections. 4-59 and 11-8 are both expressed in the calyx of the MBs, although different subgroups of Kenyon cells appear to be included in each line. OK¹⁰⁷ is a widely used MB driver. (A1-A3). Representative confocal images of pdf-lexA>lexAop-CD4::GFP¹¹/11-8-GAL4>UAS-CD4GFP¹¹⁰ brains dissected during early day (ZT2, A1), early night (ZT14, A2) and late night (ZT22, A3). (A1′-A3′) Reconstituted GFP⁺ signal is shown; the overall structure is outlined by a dashed line (encircling PDF signal) to improve visualization of the reconstituted GFP. GFP⁺ signal was observed at the 3 analyzed timepoints. (B1-B3 and B1′-B3′) Intersection between PDF and 3-86 neurons. Reconstitution signal was observed only at ZT2. (C1-C3 and C1′-C3′) A similar analysis was carried out with the 4-59 enhancer-trap line. Reconstitution was observed at ZT14. (D1-D3 and D1′-D3′) Synaptic contacts between PDF neurons and the mushroom bodies evidenced by GRASP at ZT2, ZT14 and ZT22. Arrows indicate synaptic reconstitution. Scale bar represents 10 μm unless otherwise indicated.