Neuroanatomical details of the lateral neurons of Drosophila melanogaster support their functional role in the circadian system

Abstract

Drosophila melanogaster is a long-standing model organism in the circadian clock research. A major advantage is the relative small number of about 150 neurons, which built the circadian clock in Drosophila. In our recent work, we focused on the neuroanatomical properties of the lateral neurons of the clock network. By applying the multicolor-labeling technique Flybow we were able to identify the anatomical similarity of the previously described E2 subunit of the evening oscillator of the clock, which is built by the 5th small ventrolateral neuron (5th s-LN_v ) and one ITP positive dorsolateral neuron (LN_d ). These two clock neurons share the same spatial and functional properties. We found both neurons innervating the same brain areas with similar pre- and postsynaptic sites in the brain. Here the anatomical findings support their shared function as a main evening oscillator in the clock network like also found in previous studies. A second quite surprising finding addresses the large lateral ventral PDF-neurons (l-LN_v s). We could show that the four hardly distinguishable l-LN_v s consist of two subgroups with different innervation patterns. While three of the neurons reflect the well-known branching pattern reproduced by PDF immunohistochemistry, one neuron per brain hemisphere has a distinguished innervation profile and is restricted only to the proximal part of the medulla-surface. We named this neuron "extra" l-LN_v (l-LN_v x). We suggest the anatomical findings reflect different functional properties of the two l-LN_v subgroups.

Keywords: Drosophila melanogaster; RRID: AB_141780; RRID: AB_2314041; RRID: AB_2314242; RRID: AB_2315311; RRID: AB_2315460; RRID: AB_2534096; RRID: AB_2534121; RRID: AB_2535804; RRID: AB_2535812; RRID: AB_2536186; RRID: AB_2536611; RRID: AB_300798; RRID: AB_760350; circadian clock neurons; flybow; morphology.

Figure 1

The known clock network of Drosophila melanogaster. Schematic overview shows the known arborizations of the clock neurons. The subgroups are named after their location in the brain; four small ventrolateral neurons (s‐LN_vs, red), the 5th s‐LN_v (dark violet), four large ventrolateral neurons (l‐LN_vs, brown), six dorsolateral neurons (LN_ds, orange), three lateral posterior neurons (LPN, green), and ca. 60 neurons per hemisphere in three dorsal groups (DN_1–3, lilac, cyan, blue, respectively). The figure was taken from Helfrich‐Förster et al. (2007) with permission

Figure 2

Characterization of the incompletely described Gal4‐lines. DvPdf‐G4: (a) GFP (green) and nc82 reference staining (gray) to show the overall expression pattern of the driver‐line. (b) Overview of the clock neurons that are adressed by the DvPdf‐G4 driver (indicated in red). The line drives expression in all PDF⁺ LN_vs, as well as in the 5th s‐LN_v, three CRY⁻ LN_ds, and the CRY/ITP‐coexpressing LN_d. R16C05‐G4 driver line: (c) GFP (green) and nc82 neuropil staining (gray). (d) Overview of the clock neurons that are included in the R16C05‐G4 line (indicated in red). Alongside the two CRY⁺/ITP⁻ LN_ds, two anteriorly located dorsal neurons (DN_1a) are addressed per hemisphere. R54D11‐G4 driver: (e) GFP expression (green) with nc82 neuropil staining (gray). (f) The driver includes the two only two ITP expressing clock neurons, the 5th s‐LN_v and one LN_d (indicated in red). BU, bulb; CA, calyx; LH, lateral horn; OL, optic lobe; PI, pars intercerebralis; SEZ, subesophageal zone. Scale bars = 50 µm

Figure 3

Heterogenic morphology of the large ventrolateral neurons. (a, b) Expression of mCitrine (yellow) in a single l‐LN_v and its reconstruction (orange) showing the already described morphology. (c, d) mCitrine expression (yellow) and reconstruction (magenta) of a single l‐LNv showing the so far undescribed anatomy of a subtype of l‐LNvs (“extra” l‐LN_v, l‐LN_vx) and the morphological heterogeneity within this neuronal group. (e–h) Control staining for l‐LNv heterogeneity. Flybow reporter expression driven by Pdf‐G4: (e) Single l‐LNv expressing mTorquoise in the left brain hemisphere showing the newly found morphology. The cell in the right hemisphere (asterisk) is bleeding through from another channel, but all visible projections are stemming from the l‐LN_vx. (f) Two l‐LN_vs in the same brain expressed mCitrine (yellow), one cell in each hemisphere, showing the already described projection pattern. (g) Anti‐PDF staining showing the well‐characterized projection pattern of the PDF⁺ LN_vs, confirming the structural integrity of the network. (h) Merge of the previous channels. (i, j) Projections of the l‐LN_vs are extensively invading the serpentine layer of the medulla. The majority of l‐LN_v neurites run on the surface of the medulla (i; arrow in j), but a remarkable proportion is invading the serpentine layer (j). The boundary between the inner and outer layers can be seen in the dorsal and distal area of the medulla (indicated with arrowheads in j). (k) Schematic representation of the initial branching pattern of l‐LN_vs. The primary projection (red) runs ventrally from the soma (black sphere) along the medial edge of the ipsilateral medulla and forms the ventral elongation of the accessory medulla (AME_vel). A secondary fiber (blue) branches off from the initial projection, runs through the posterior optic commissure (POC) and is thereby connecting both hemispheres, to eventually arborize onto the surface of the contralateral ME. The fiber network on the ipsilateral ME is built by the branches of a third side‐projection (orange) which separates from the fiber that runs through the POC. Scale bars = 50 μm

Figure 4

Projection pattern of the 5th s‐LNv and the ITP expressing LNd. (a) Overview of the 5th s‐LN_v expressing mCherry (magenta) and nc82 neuropil staining (gray). The part that was cut out for the 3D view (e, f) is shown by the dashed square. (b) Scheme of the 5th s‐LN_v. (c‐d) Magnification. (e, f) Anterior‐lateral (e) and posterior‐lateral (f) view of the 5th s‐LN_v. The optic lobe of the left hemisphere was partly cut out (as indicated in a) for a better view on the soma and the initial branching. (g–l) Morphology of the CRY⁺/ITP⁺ LN_d. (g) Amplified mCherry expression (magenta) with nc82 neuropil staining (gray). The dashed square indicates the section that was cut out for the 3D view in (k) and (l). (h) Schematic overview of the same neuron. (i, j) Magnification. (k, l) Anterior‐lateral (k) and posterior‐lateral (l) view of the neuron and its location in the brain. The dorsal part of the left optic lobe was cut out (indicated in g) for improved visibility of the projections running toward the optic neuropils. Orientation of the brain (e, f and k, l) is declared by the coordinate system. a, anterior; d, dorsal; x, lateral axis. ME, medulla; LO, lobula; LOP, lobula plate; CA, calyx; AME, accessory medulla; PLP, posterior lateral protocerebrum; LH, lateral horn; SCL, superior clamp; ICL, inferior clamp; SLP, superior lateral protocerebrum; SMP, superior medial protocerebrum; MDC, middle dorsal commissure; SMP`, superior medial protocerebrum of the contralateral hemisphere. Scale bars = 50 μm

Figure 5

Characteristic anatomical features of the ITP expressing lateral clock cells. 5th s‐LN_v single cell projection pattern rotated and viewed from different angles in 60° steps (upper two panels). In the plane view (0°) the main branch was labeled (red) and characteristic branches were numbered for further description (see maintext). For easier orientation, the dorsoventral midline is indicated (dashed blue line) and landmark fiber bundles are implied (POC, posterior optic commissure). Included neuropil structures are shown in dark green. The location of the cell body is highlighted in purple (rAVLPl). In the dorsal brain, the main projection reaches into the contralateral hemisphere via the middle dorsal commissure (MDC). Rotated ITP⁺ LNd single cell projection pattern (lower panels). The dorsal and ventral main projections were labeled (red and light green, respectively) and characteristic branches were numbered for detailed description (see maintext). The dorsoventral midline (dashed blue line) and landmark fiber bundles are indicated (AOT, POC). Like the 5th s‐LNv, the LN_ds' dorsal main projection is crossing the dorsoventral midline via the MDC (blue). AME, accessory medulla; AOT, anterior optic tract; rAVLPl; cell body rind lateral to the anterior ventrolateral protocerebrum; SCL, superior clamp; PED, mushroom body peduncle; rLHla, cell body rind lateroanterior to the lateral horn

Figure 6

Morphology of the two sNPF⁺/CRY⁺ LN_ds. (a) Depiction of one of the two CRY⁺ but ITP^‐ LN_ds labeled with mCherry antibody (magenta) shown together with a nc82 neuropil staining (gray). (b) Schematic overview. (c, d) Magnification. (e, f) Anterior‐lateral (e) and posterior lateral (f) view on the 3D brain. The orientation is indicated by the coordinate system. Rotated sNPF⁺/CRY⁺ LN_d (lower panel). The dorsal and ventral main branches are labeled (red and light green, respectively) and characteristic branches were numbered for further description in the maintext. The dorsoventral midline is indicated (dashed blue line), and landmark fiber bundles are implied (AOT) for easier orientation. Additional information on neuropil structures are shown in dark green, whereas the location of the cell body is highlighted in purple (rLHla). The dorsal main projection contibutes to the MDC (blue) and passes into the contralateral hemisphere. a, anterior; d, dorsal; x, lateral axis. AOTU, anterior optic tubercle; AOT; anterior optic tract; ME, medulla; LO, lobula; LOP, lobula plate; CA, calyx; PLP, posterior lateral protocerebrum; LH, lateral horn; SLP, superior lateral protocerebrum; SIP, superior intermediate protocerebrum; SMP, superior medial protocerebrum; MDC, middle dorsal commissure; SMP`, superior medial protocerebrum of the contralateral hemisphere; rLHla, cell body rind lateroanterior to the lateral horn. Scale bars = 50 μm

Figure 7

Neuroanatomy of the three CRY‐negative LN_ds. (a) Exemplary overview of one of the three CRY⁻ LN_ds, labeled with an antisera against mCherry (magenta) and costained for the neuropils with nc82 (gray). (b) Scheme of a CRY⁻ LN_d. (c, d) Magnification. (e, f) Anterior‐lateral (e) and posterior‐lateral (f) view. Projection pattern rotated and viewed from different angles in 60° steps (lower panel). Characteristic branches are numbered for further description in the maintext and landmark fiber bundles are implied in orange (AOT). Neuropil structures are shown in dark green and the location of the cell body is highlighted in purple (rLHla). The projections do not cross the dorsoventral midline and are restricted to the ipsilateral brain hemisphere (see also a and d). a, anterior; d, dorsal; x, lateral axis. AOT, anterior optic tract; ME, medulla; LO, lobula; LOP, lobula plate; CA, calyx; PLP, posterior lateral protocerebrum; LH, lateral horn; SLP, superior lateral protocerebrum; SIP, superior intermediate protocerebrum; SMP, superior medial protocerebrum; MDC, middle dorsal commissure; SMP`, superior medial protocerebrum of the contralateral hemisphere; rLHla, cell body rind lateroanterior to the lateral horn. Scale bars = 50 μm

Figure 8

Reconstructions of representative single cell projections registered to the Janelia Farm standard brain (Jenett et al., 2012) and innervation map. Neuronal subgroups that build different E‐oscillator subunits either show the same (E1 and E3) or highly comparable innervation patterns, even though they belong to different clock neuron subgroups, like the neurons of E2. (a–d) Exemplary reconstructions of the single cell projection patterns of the lateral clock neurons that comprise the E‐oscillator. (a) Two LN_ds per brain hemisphere express the circadian photoreceptor Cryptochrome (CRY) and the short neuropeptide F (sNPF). The two neurons comprise the E1‐oscillator subunit and are not distinguishable from each other in respect to their morphology or neurochemical content. (b, c) The E2‐oscillator is built by two neurons that belong to different clock neuron subclusters, and their cell bodies are located in discrete areas of the brain. The LN_d and the 5th s‐LN_v both co‐express CRY and ITP and show a similar innervation pattern (see f), although they belong to different neuronal subgroups (ventrolateral neurons and dorsolateral neurons, respectively). There is only one cell of each of those neurons per brain hemisphere. (d) The E3‐oscillator subunit consists of three CRY⁻ LN_ds per brain hemisphere that are morphologically equal. The only criteria for discrimination is the expression of neuropeptide F (NPF) by two of the three E3 cells (Johard et al., 2009). (e) Superposition of single E‐cell subtypes onto the Janelia Farm standard brain. (f) Innervation map based on the projection pattern of individually analyzed lateral clock neurons (see Table 3). ME, medulla; AME, accessory medulla; PLP, posterior lateral protocerebrum; ICL, inferior clamp; SCL, superior clamp; AOTU, anterior optic tubercle; LH, lateral horn; SLP, superior lateral protocerebrum; SIP, superior intermediate protocerebrum; SMP, superior medial protocerebrum; SMP`, contralateral SMP; SIP`, contralateral SIP; SLP`, contralateral SLP

Figure 9

Post‐ and presynaptic sites of the ITP expressing LN_d together with the 5th s‐LN_v. (a) UAS‐nSyb expression (green) of R54D11‐G4/pdf‐G80 brains shows putatively presynaptic sites and (b) UAS‐DenMark (magenta) driven by the same Gal4/Gal80 combination reveals the postsynaptic projections of the ITP⁺ LN_d and the 5th s‐LN_v. (c, d) Schematic overview of the 5th s‐LNv's (c) and the ITP⁺ LN_d's (d) polarity with presynaptic sites in green and postsynaptic sites in magenta. The fascicle which runs from the posterior lateral protocerebrum (PLP) to the lobula (LO) was exclusively labeled with the postsynaptic marker (left arrow in both overviews). Both cells possess characteristic postsynaptic fibers that are only present in the respective cell type (right arrow in both overviews). ITP, ion transport peptide; LH, lateral horn; SLP,superior lateral protocerebrum; SMP, superior medial protocerebrum; CA, calyx; LOP, lobula plate; ME, medulla. Scale bars = 50 μm

Figure 10

Post‐ and presynaptic sites of the ITP‐negative LN_ds. (a) Expression of R16C05‐G4/UAS‐nSyb (green) to reveal putative presynaptic arborizations of the CRY⁺ LN_ds together with the DN_1a. (b) R16C05‐G4/UAS‐DenMark expression (magenta) in the postsynaptic branches of the CRY⁺ LN_ds and the DN_1a. The soma of a non‐clock cell can be seen at the lateral edge of the lateral horn (asterisk), but the marker expression in the neurites was too weak to interfere with the identification of clock cell projections. (c) Schematic overview of the polarity of CRY⁺ LN_ds with postsynaptic sites in magenta and presynaptic sites in green. The projection on the surface of the lateral horn (LH) expressed the presynaptic marker (green arrows), but the majority of the arborizations in the ipsilateral superior brain regions could only be seen with the postsynaptic marker. (d–f) Post‐ and presynaptic sites of the CRY⁻ LN_ds. (d) UAS‐nSyb (green) expression shows putative presynaptic sites in the brains of DvPdf‐G4/cry‐G80 flies and (e) UAS‐DenMark (magenta) revealed the postsynaptic arborizations. Cry‐G80 did not sufficiently suppress the reporter expression in the PDF⁺ LN_vs, but worked in the ITP⁺ LNd and the 5th s‐LN_v. (f) Schematic overview of the post‐ and presynaptic sites of the CRY⁻ LN_ds in magenta and green, respectively. CRY, cryptochrome; PLP, posterior lateral protocerebrum; AOTU, anterior optic tubercle; SLP, superior lateral protocerebrum; CA, calyx; LO, lobula; LOP, lobula plate; ME, medulla. All scale bars = 50 μm

Figure 11

Comparison of the estimated cell diameters of clock neurons. A one way ANOVA with post hoc Bonferroni correction was performed for statistical testing. Contrary to the current nomenclature the 5th s‐LN_v shows the third largest cell diameter, surpassed in size only by the l‐LN_vs and the ITP⁺ LN_d (not significant) and it is significantly larger than the PDF⁺ s‐LN_vs. Significant differences are indicated with different letters. The same letter means that there was no statistical disparity in cell diameter between the cell types. All indicated differences were highly significant with p < .01. CRY, cryptochrome; ITP, ion transport peptide. Scale bar = 50 μm

Figure 12

Comparison of the soma morphology of the E‐cells. In contrast to the CRY⁻ LN_ds, the somata of all the CRY expressing E‐cells possess small membrane‐appendages. The same appendages can be seen with DenMark expression, suggesting that these varicosities are likely postsynaptic. CRY, cryptochrome; ITP, ion transport peptide. Scale bars = 10 μm