Development and ultrastructure of <i>Drosophila</i> larval VNC.

<p>The development process of <i>Drosophila</i> larval VNC (<b>A–I</b>). VNC appearance and length are not significantly different in the periods of L1–2 (<b>A</b>), L1–12 (<b>B</b>), L1–22 (<b>C</b>) and L2–2 (<b>D</b>). The length of VNC in the L2–12 period (<b>E</b>) was longer than the L2–2 period (<b>D</b>), and once again with no obvious difference between L2–12 period and L2–22 period (<b>F</b>). The shape and the length of VNC display an obvious change during the L3–2 (<b>G</b>), L3–12 (<b>H</b>) and L3w (<b>I</b>) periods. White arrow indicates the brain, and black arrow indicates VNC. The ultrastructure of neuropils at low magnification in L1–2 period (<b>J</b>), L1–12 period (<b>K</b>) and L3w period (<b>L</b>). The VNC neuropil is wrapped by inner glial cells, and they were filled with nerve fibers and neurites (<b>J</b>–<b>L</b>). The size of neurites and neuropil in L1–2 period (<b>J</b>) are not obviously different than the L1–12 period (<b>K</b>), but size of neurites and neuropil in the L3w period (<b>K</b>) is bigger than L1–2 period (<b>J</b>) and L1–12 period (<b>K</b>). Most of the neurites are in S phase and T phase in L1–2 period (<b>J</b>) and L1–12 period (<b>K</b>). The black triangle indicates the neuronal soma, the white triangle indicates the inner glial cell and the thick arrow indicates nerve fiber. The large black box is enlarged from the small white box in the same diagram, and indicates the CV_I neurite. Scale Bar A–I: 50 µm; J–K: 5 µm; L: 10 µm.</p

Identification four classes of neurites in <i>Drosophila</i> VNC.

<p>Clear vesicle (CV) neurite contains clear vesicles at L3w period and the CV_I is light-colored and globular (<b>A</b>); Dense-core vesicle (DV) neurite contains dark dense-core vesicles (<b>B</b>, <b>E</b> and <b>I</b>). DV_I neurite contains the similar size of dark dense-core vesicles (<b>E</b>, <b>I</b>), and DV_II neurite contains bigger and different size dark dense-core vesicles (<b>B</b>). Mixed vesicle (MV) neurite contains both clear and dense-core vesicles (<b>C</b>, <b>J</b>). Large vesicle (LV) neurite contains large and spherical translucent vesicles (<b>D</b>, <b>F</b> and <b>H</b>). All the DV neurites, MV neurites and LV neurites are globular (<b>A</b>–<b>I</b>). In the same diagram, the large black box is enlarged from the small white box. <b>G</b> indicates the DV vesicles in an axon at L3w period. Triangle indicates synapse, arrow indicates big size dense-core vesicle, asterisk indicates the central region of CV neurite and Mi indicates mitochondria. Scale Bar: 500 nm.</p

The model of CV_I neurite development.

<p><b><i>P</i></b> indicates P phase, <b><i>S</i></b> indicates S phase, <b><i>T</i></b> indicates S phase, <b><i>B</i></b> indicates B phase and <b><i>M</i></b> indicates M phase. Note: the neurites in P phase, S phase, and T phase are globular, while the neurites in B phase are bifurcated or dendritic, and the neurites in M phase return to globular again. There are big dark synaptic vesicles in S phase, T phase, and B phase, and few big dark synaptic vesicles in M phase. The new neurites in P phase bubbled continuously along the axon throughout the larval stage.</p

Main axons expand and form irregularly CV_I neurites.

<p><b>A</b> shows the longitudinal section of a nerve fiber that is made up of the main axons whose diameter is roughly similar to some neurites in B phase; <b>B</b> shows the cross-section of a nerve fiber that is made up of main axons; <b>C</b> shows one of the main axons is developing into S phase, and another is developing into B phase in the cross-section. <b>D</b> shows one of main axons is developing into S phase in the longitudinal section. The main axon forms into irregularly shaped neurites in B phase and three branches in L1–2 period (<b>E</b>) and L1–22 period (<b>F</b>), with some big dark synaptic vesicles. <b>H</b> shows the main axon forms an irregularly shaped neurite and two branches in L3–12 period, with some big dark synaptic vesicles. <b>G</b> shows the main axon forms irregularly shaped neurites in L3w period, with some big dark synaptic vesicles and typical T-bar structures. <b>I</b> diagram shows another main axon forms irregularly shaped neurites in L3w period, with some typical T-bar structures but few big dark synaptic vesicles. a indicates axon, <b><i>S</i></b> indicates S phase, <b><i>B</i></b> indicates B phase and <b><i>M</i></b> indicates M phase. Arrow indicates the big dark synaptic vesicles, triangle indicates synapse, asterisk indicates the branch and Mi indicates mitochondria. In the same diagram, the large black box is enlarged from the small white box. Scale Bar A: 1000 nm, B–F, H–I: 500 nm; G: 200 nm.</p

The new neurites produce via budding at the end of axon and bubbling along the axon.

<p><b>A</b>–<b>E</b> shows the new neurites bud at the end of axons in different phases, and the terminal of the fine axon is connected to intumescent neurite. <b>A</b>, <b>C</b> and <b>E</b> show the neurites in T phase at early stage 17 embryo (<b>F</b>), L1–2 period (A) and L3w period (<b>C</b>); <b>B</b>–<b>C</b> show the neurites in B phase in L1–12 period (<b>B</b>) and L3w period (<b>C</b>); <b>D</b> shows the neurite in M phase in L3w period. <b>E</b>–<b>I</b> shows neurites bubbling along the axons, and three neurites in B phase grow on one axon in L1–2 phase (G) and L2–22 phase (<b>H</b>). <b>I</b> diagram shows two neurites in B phase bubbling along the axon in L3–2 period. <b>E</b> shows the nascent neurite in P phase bubbling along the axon. <b>F</b> shows two axons beside one neurite in T phase. In <b>G</b> diagram, the large black box is enlarged from the small white box, and the large black box is enlarged from the small white box. <b><i>T</i></b> indicates T phase, <b><i>B</i></b> indicates B phase and <b><i>M</i></b> indicates M phase. White triangle indicates synapse, black long triangle indicates the neurite in P phase, arrow indicates the big dark synaptic vesicles, asterisk indicates the axon and Mi indicates mitochondria. Scale Bar A–B, E: 200 nm; C, F: 2000 nm; D: 500 nm; G–I: 1000 nm.</p

The CV_I neurites are divided into five developmental phases.

<p>In P phase (<b>A</b>–<b>B</b>, <b>Q</b>), there are no synapses, microtubules, or synaptic vesicles in the neurite. In S phase (<b>C</b>–<b>D</b>, <b>R</b> and <b>T</b>), there are some microtubules, mitochondria, or a few small or big dark synaptic vesicles, but no synapse in the neurite. In T phase (<b>E</b>–<b>F</b>, <b>L</b> and <b>S</b>), the synapses have immature T-bar structures, a few synaptic vesicles gathered around the small T-Bar structure, and microtubules disappeared in the neurite. In B phase (<b>G</b>–<b>K</b>, <b>T</b>–<b>U</b>), the neurites look bifurcated or dendritic, the T-bar structure is incomplete, and the two different diameters of synaptic vesicles are still present. In M phase, (<b>M</b>–<b>O</b>, <b>V</b>) there are a lot of commensurate synaptic vesicles cluster around the typical T-bar structure, and few big dark synaptic vesicles; <b>P</b> shows the NMJ T-bar structure. All the neurites in P phase (<b>Q</b>), S phase (<b>R</b>, <b>T</b>), T phase (<b>S</b>), B phase (<b>T</b>–<b>U</b>) and M phase (<b>V</b>) can be observed simultaneously at L3w period, and there are no synaptic vesicles in the center of the big neurites (<b>Q</b>–<b>V</b>). In the same diagram, the large black box is enlarged from the small white box, and <b>N</b> is the magnification of the <b>M</b> diagram box. <b><i>P</i></b> indicates P phase, <b><i>S</i></b> indicates S phase, <b><i>T</i></b> indicates T phase, <b><i>B</i></b> indicates B phase and <b><i>M</i></b> indicates M phase. Triangle indicates synapse, thick arrow indicates the big dark synaptic vesicle and thin arrow indicates microtubule (B–D), and Mi indicates mitochondria. Scale Bar A–C: 100 nm; D–F, H–I, K–L, O–P: 200 nm; G, J, M: 500 nm.</p

The CV_II neurites are divided into five developmental phases.

<p>In P phase (<b>A</b>), there are not synapses, microtubules, or synaptic vesicles in the neurite. In S phase (<b>B</b>), there are some microtubules, mitochondria, or different sizes of synaptic vesicles, but no synapses. In T phase (<b>C</b>–<b>D</b>), there are residual microtubules and a greater variety in the size of synaptic vesicles in the neurite; the synapse is immature, and there is no T-bar structure formation. In B phase (<b>B</b>, <b>E</b>–<b>L</b>), the neurites look bifurcated or dendritic, and the two different diameters of synaptic vesicles remain present. In M phase, there are dark cytoplasm, typical T-bar structures and a lot of commensurate synaptic vesicles cluster around it, but the appearance of the neurite remain bifurcated or dendritic, and have several branches around it (<b>M</b>–<b>N</b>). In embryo stage, the synapses have not T-bar structures (<b>F</b>), but have the immature T-bar structures and dark cytoplasm throughout the larval stage (<b>G</b>–<b>L</b>) in B phase. In the same diagram, the large black box is enlarged from the small white box, and <b>H</b> is the magnification of the <b>G</b> diagram box. Triangle indicates synapse, thick arrow indicates the big dark synaptic vesicles, thin arrow indicates microtubules, asterisk indicates the axon and black long triangle indicates the nascent branch of neurite in B phase (<b>B</b>). <b><i>P</i></b> indicates P phase, <b><i>S</i></b> indicates S phase, <b><i>T</i></b> indicates T phase, <b><i>B</i></b> indicates B phase, <b><i>M</i></b> indicates M phase and Mi indicates mitochondria. Scale Bar A: 100 nm; B–H, K–M: 200 nm; I: 500 nm.</p

The preexisting neurite buds, divides and produces the CV_I neurites.

<p><b>A</b> shows the neurite in B phase issue five branches (asterisk indicates) at L1–2 period, and two of the branches (upper right corner) have no synaptic vesicle. <b>B</b>–<b>C</b> shows the neurite issues two branches in B phase at L2–22 period. <b>D</b> shows the neurite issues three branches in B phase at L3–2 period. <b>E</b>–<b>K</b> shows the preexisting neurite divides and produces a new neurites. <b>F</b>–<b>I</b> shows the big furrow and some microtubules between the preexisting neurite and the new neurite. J shows the furrow is narrow, with no microtubules visible in it. The right part of <b>K</b> shows the new neurite developing into M phase, and the uniform distribution of synaptic vesicles around the neurite. <b>E</b> shows the neurite in S phase divides and produces the new neurite at L3w period. In the same diagram, the large black box is enlarged from the small white box. Triangle indicates synapse, thick arrow indicates the big dark synaptic vesicle, thin arrow indicates microtubule, asterisk indicates the branch of B neurite and Mi indicates mitochondria. <b><i>S</i></b> indicates S phase, <b><i>T</i></b> indicates T phase, <b><i>B</i></b> indicates B phase and <b><i>M</i></b> indicates M phase. Scale Bar A, E–F: 200 nm; B–D, G–K: 500 nm.</p

The model of CV_II neurite development.

<p><b><i>P</i></b> indicates P phase, <b><i>S</i></b> indicates S phase, <b><i>T</i></b> indicates S phase, <b><i>B</i></b> indicates B phase and <b><i>M</i></b> indicates M phase. Note: There are microtubules in the T phase of CV_II neurite (C), the shape is bifurcated or dendritic in the B phase (D–E) and the M phase of CV_II neurite (F), but it is globular in the M phase of CV_I neurite.</p

Characterization of vesicles and neurites in different developmental periods.

<p><b>A–D</b> shows the quantifications of vesicles’ diameters of in neurites. <b>A</b> shows the size of vesicles in four types of neurites at L3w period. <b>B</b> shows the diameter of big dark synaptic vesicles in developmental CV_I neurites in the first instar larvae. The big dark synaptic vesicles in T phase are smaller in L1–12 period when compared to L1–2 period (P = 0.035). <b>C</b> shows the big dark synaptic vesicles do not show statistically significant difference at developmental periods, and among the first instar larval stage, the second instar larval stage and third instar larval stage, however, there is statistically significant difference between the first instar larval stage and the second instar larval stage (P = 0.043). <b>D</b> shows no difference of total big dark synaptic vesicles between CV_I and CV_II neurites in the whole instar larval stage. <b>E–G</b> shows the quantifications of the density and percentage in the first instar larval CV neurites. <b>E</b> shows the CV neurites decreases in P and S phase, but increases in B phase from L1–2 period to L1–22 period. However, the CV neurites, in T phase, increases significantly from L1–2 period to L1–12 period, but decreases from L1–12 period to L1–22 period. <b>F</b> shows percentage of CV neurites in P, S, T, B phase. From L1–2 period to L1–12 period, the percentage of S phase is the highest. At L1–22 period, the percentage of S and B phase are the highest. <b>G</b> shows the number of total CV neurites including P, S, T and B phase decreases from L1–2 period to L1–12 period, and from L1–12 period to L1–22 period. <b>S</b> indicates S phase, <b>T</b> indicates S phase, <b>B</b> indicates B phase. N = Number of samples. Error bar indicates SEM, t test. *p<0.05, **p<0.01, ***p<0.001, ns = non-significant.</p