Glial Processes at the Drosophila Larval Neuromuscular Junction Match Synaptic Growth

Glia are integral participants in synaptic physiology, remodeling and maturation from blowflies to humans, yet how glial structure is coordinated with synaptic growth is unknown. To investigate the dynamics of glial development at the Drosophila larval neuromuscular junction (NMJ), we developed a live imaging system to establish the relationship between glia, neuronal boutons, and the muscle subsynaptic reticulum. Using this system we observed processes from two classes of peripheral glia present at the NMJ. Processes from the subperineurial glia formed a blood-nerve barrier around the axon proximal to the first bouton. Processes from the perineurial glial extended beyond the end of the blood-nerve barrier into the NMJ where they contacted synapses and extended across non-synaptic muscle. Growth of the glial processes was coordinated with NMJ growth and synaptic activity. Increasing synaptic size through elevated temperature or the highwire mutation increased the extent of glial processes at the NMJ and conversely blocking synaptic activity and size decreased the presence and size of glial processes. We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj. Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ

Distribution of acetylcholine and catecholamines in fish gills and their potential roles in the hypoxic ventilatory response

Carotid body glomus cells in mammals contain a plethora of different neurochemicals. Several hypotheses exist to explain their roles in oxygen-chemosensing. In the present study we assessed the distribution of serotonin, acetylcholine and catecholamines in the gills of trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) using immunohistochemistry, and an activity-dependent dye, Texas Red hydrazide (TXR). In fish the putative oxygen sensing cells are neuroepithelial cells (NECs) and the focus in recent studies has been on the role of serotonin in oxygen chemoreception. The NECs of trout and goldfish contain serotonin, but, in contrast to the glomus cells of mammals, not acetylcholine or catecholamines. Acetylcholine was expressed in chain and proximal neurons and in extrinsic nerve bundles in the filaments. The serotonergic NECs did not label with the HNK-1 antibody suggesting that if they are derived from the neural crest, they are no longer proliferative or migrating. Furthermore, we predicted that if serotonergic NECs were chemosensory, they would increase their activity during hypoxia (endocytose TXR), but following 30. min of hypoxic exposure (45. Torr), serotonergic NECs did not take up TXR. Based on these and previous findings we propose several possible models outlining the ways in which serotonin and acetylcholine could participate in oxygen chemoreception in completing the afferent sensory pathway.12 page(s

Glial processes were expanded in the highwire mutation.

<p>A–C) Live NMJs from F3 larvae with glial processes labeled with repo>CD8GFP (green) and boutons immunolabeled with α-HRP (magenta). NMJs are shown from <i>hiw</i> larvae raised at 18°C (A), 25°C (B) and 30°C (C). Glial areas: A  = 26.7 µm²; B  = 26.2 µm²; C  = 16.7 µm². Scale bars, 15 µm. D–H) Grayscale images showing details of the glial processes from the corresponding boxed regions in A–C. Panels were digitally scaled 200%. Glial processes extended away from the NMJ across the muscle surface (arrowheads) or tracked along the center of the bouton area (arrows). Filopodial-like processes were also frequently observed (open arrowheads). I–J) Statistical comparison of the glial process areas from F3 <i>hiw</i> larvae (<i>hiw</i>; red) compared to wild-type (wt; blue) larvae raised at 18°C (circles), 25°C (squares) or 30°C (diamonds). I) The mean area of glial processes was significantly larger in <i>hiw</i> mutants than wild-type at 18°C and 25°C (P<0.0001) but not 30°C. Black symbols represent those NMJs with glial processes that stopped at the motor axon end. J) The mean ratio of the GP area to neuronal (α-HRP) area was not significantly different between <i>hiw</i> and wild-type larvae at any rearing temperature.</p

Increased rearing temperature expanded the perineurial glial processes.

<p>A–D) Live NMJs from F3 larvae with glial processes labeled using 46F>CD8-GFP (green), axons plus boutons immunolabeled with anti-HRP (red) and the SSR labeled with ShCter-DsRed (blue). Scale bars, 15 µm. The boxed regions (i) were digitally scaled 400% and all three channels shown in grayscale. A–B) NMJs from larvae raised at 18°C. Ai–Bi) Glial processes were small and most stopped at the axon end prior to the proximal boutons in the region corresponding to the end of the blood-nerve barrier (arrowheads). Glial areas: A = 15.7 µm²; B = 14.0 µm² C–D) NMJs from larvae grown at 30°C. Ci–Di) Glial processes were more extensive and processes occupied a central channel along the synapse (arrows), plus extended across the muscle away from the NMJ (concave arrowhead). Glial areas: C = 16.0 µm²; D = 20.1 µm² E–G) Glial membrane areas were measured and compared to neuron/bouton area (anti-HRP) and SSR area at 18°C, 25°C and 30°C. E) The mean area of glial processes (green) was significantly higher in larvae reared at 30°C (diamonds) than 18°C (circles) or 25°C (squares) (P<0.0002). Black symbols represent NMJs with glial processes that stopped at the motor axon end prior to the first bouton in the region defined as the terminus of the blood-nerve barrier. F) The mean ratio of glial process (GP) to neuron/bouton area (anti-HRP)(blue) was significantly greater at 30°C (diamonds) compared to 18°C (circles) and 25°C (squares) (P<0.0125). G) The mean ratio of GP to SSR area (magenta) was significantly greater for larvae reared at 30°C (diamonds) compared to 18°C (circles) and 25°C (squares) (P<0.0302).</p

Live imaging revealed that glial processes extended into the NMJ.

<p>A, B) Live larval NMJs labeled for all three components of the tripartite synapse. Membrane targeted GFP (CD8-GFP) was expressed in glia using different GAL4 drivers in live NMJs where the axons and boutons were live labeled with an anti-HRP antibody (α-HRP, red) and the SSR labeled with ShCter-DsRed (blue). Each panel is a compressed stack of the entire NMJ (12–14 µm). Scale bars, 15 µm. A) repo-GAL4 driving UAS-CD8-GFP in all glia (repo>CD8-GFP, green) labeled the processes in the NMJ from both the subperineurial and perineurial glia. B) 46F-GAL4 driving UAS-CD8-GFP in only the perineurial glia (46F>CD8-GFP, green) labeled similar glial structures seen with the repo-GAL4 driver. C, D) Membrane targeted RFP was expressed in the perineurial glia using 46F-GAL4 (46F>CD-RFP, red) in relationship with known markers in fixed peripheral nerves. Inserts are cross-sections of the nerve at the points marked by the dashed lines. Scale bars, 15 µm. C) 46F>CD8-RFP (red) in the perineurial glia was adjacent to the secreted neural lamella labeled with Perlecan-GFP (green). The image represents a single 0.2 µm longitudinal section through the center of the nerve. D) 46F>CD8-RFP (red) in the perineurial glia is exterior to the expression of GFP-tagged Neurexin IV (NrxIV-GFP, green) in the underlying subperineurial glia. A projection of the entire stack is shown. E) Jupiter-GFP labeled perineurial glia extend over the subperineurial glia labeled using Gli>CD8-RFP as axons extend from the main nerve to their target muscles (arrows). F) 46F>CD8-RFP labeled perineurial glia extend over the subperineurial glia labeled using NeurexinIV-GFP (Nrx-GFP) as axons (α-HRP, blue) extend from the main nerve to their target muscles (arrow).</p

The blood-nerve barrier does not extend beyond the motor axon.

<p>A–C) Live NMJ from a F3 larva reared at 30°C. The glial membrane processes were labeled using repo>CD8-GFP (green) and the SSR labeled using ShCter-DsRed (blue). A 10 kDa fluorescent dextran (red) was used to test for the presence of a permeability barrier. Glial area  = 13.77 µm². Scale bar, 15 µm. A) A single Z-slice from a focal plane at the surface of the NMJ showed dextran permeation into the synaptic ^region. The motor nerve projection is marked (asterisk) between muscles 6 and 7, and showed dye exclusion from the motor axons. B) The boxed region in panel A was digitally scaled by 200%. The fluorescent dextran pooled around the SSR even with the close association of the glial processes (arrows). C) The boxed region in A was digitally scaled by 200%. Fluorescent dextran was excluded along the axon (between the arrows) where the GFP tagged glial membrane was present and demarks the regions where the blood-nerve barrier is found.</p

Statistics for glial, neuronal, SSR areas and ratios.

<p>Mean and standard deviation (SD) values for measured areas of glial processes (CD8-GFP), neurons (anti-HRP immunolabeling) and SSR (ShCter-DsRed). Glial processes were measured from the terminal region of axon as it enters the synapse. The ratios of glial process area to neurons or SSR are indicated. Replicate numbers of NMJs and larvae are indicated for each experimental protocol. F3, feeding third instar larvae. W3, wandering third instar larvae. WT, wild-type. Fixed, fixed larvae; all other larvae were live. T0, T60; 0 and 60 minute time points. 30→18°C, temperature down-shift from 30 to 18°C after hatching. 18→30°C, temperature up-shift from 18 to 30°C after hatching. Areas are in µm².</p

Changes in motor activity and synaptic growth affect the presence of glial processes at the larval NMJ.

<p>Live NMJs from F3 larvae with glial processes labeled using repo>CD8GFP, neurons immunolabeled with anti-HRP and the SSR labeled with ShCter-DsRed. A) TTX treatment: comparison of glial process areas (green) and SSR areas (blue) from control (C, diamonds) or TTX treated (TTX, hexagons) larvae raised at 30°C. TTX significantly reduced the mean area of glial processes relative to control animals (P<0.0001) but had no effect on the mean SSR area. Black symbols represent NMJs with glial processes that stopped at the motor axon end just prior to the first bouton. The mean ratio of glial process to SSR area (magenta) was significantly reduced in TTX treated larvae (hexagons) compared to control larvae (diamonds) (P<0.0004). B) F3 compared to W3: comparison of glial (green) and SSR areas (blue) from feeding third instar (F3, circles) or wandering third instar (W3, squares) larvae raised at 25°C. Mean glial areas and mean SSR areas were not significantly different between F3 and W3. The mean ratio of glial to SSR area (magenta) in F3 larvae (circles) was comparable to the ratio in W3 larvae (squares). C) Temperature shift treatment: comparison of glial areas from larvae reared continuously at 18°C (blue circles), shifted from 18°C to 30°C (pink circles), shifted from 30°C to 18°C (purple diamonds) and raised continuously at 30°C (red diamonds). The mean glial area from larvae transferred from 18°C to 30°C was significantly smaller than in larvae grown continuously at 30°C (P<0.0002), but the same size as larvae grown at 18°C. The mean ratio of glia to SSR area at 30°C constant rearing temperature was significantly different from all other protocols (P<0.002).</p

Glial processes at the NMJ had a wide range of morphologies.

<p>A–D) Live NMJs with the perineurial glial membranes labeled using 46F>CD8-GFP (green), neurons immunolabeled with anti-HRP (red) and the SSR labeled using ShCter-DsRed (blue). The panels are projections of the entire NMJ (12–14 µm thick) from F3 larvae grown at 25°C. Boxed inserts (i–viii) were digitally scaled 400%. Scale bars, 15 µm. A) A NMJ where the glial processes end with blunt processes at the axon terminus just prior to the first proximal bouton (Ai, Aii; arrowheads). Glial processes also contacted discrete regions of each bouton (Aii; arrows). Glial area  = 13.7 µm². B) A NMJ where the glial processes extended across the muscle surface (Bi, Bii; concave arrowheads) or track along a channel over the bouton and SSR (Bi; arrows). Glial area  = 20.7 µm². C) A W3 NMJ where glial processes had extensive projections into the NMJ and tracked along the center of the synaptic region (Ci, Cii; arrows). Glial area  = 16.85 µm². D) A NMJ where glial processes formed node-like processes embedded in the muscle (concave arrowhead). These processes extended away from the NMJ and were not associated with boutons or SSR. Glial area  = 18.6 µm². D.i–D.iv) Inserts show the node-like glial processes (double arrowheads) embedded in the muscle imaged over a series of single 0.2 µm focal planes. D.v–D.viii) The boxed region in panel D was re-sectioned left to right (v to viii) on the YZ axis. The glial nodes (double arrowheads) were spherical and approximately 3 µm thick. The top of each panel is the approximate coverslip position.</p