Publication
Dissection of glial contributions to neuronal structure and function in Drosophila melanogaster
| Summary: | Synaptic plasticity is a process by which neurons remodel their structure and/or molecular composition of synapses. This mechanism underlies nervous system connectivity and allows for cognition and memory formation. The Drosophila peripheral nervous system has been extensively used to characterize mechanisms of plasticity. The peripheral nerves are wrapped by three glial layers: the innermost wrapping glia (WG), middle layer of subperineurial glia (SPG) and the outermost perineurial glia (PG). The SPG and PG subtypes extend multiple membranous processes (namely lammelipodia-like structures, gliopods and gliobuls) into the larval neuromuscular junctions (NMJs). The NMJ synapse is established between motor neuron presynaptic terminals that innervate muscle cells and enables robust analysis of activity-induced structural plasticity. On motor terminals of 3rd instar NMJs, new boutons are added to the existing NMJ in response to developmental cues and in an activity-dependent manner. New boutons can be induced acutely through stimulation protocols based on patterned high K+ stimulation, or chronically by raising larvae at 290C, which increases larval locomotion and therefore activity. SPG at the NMJ has been previously shown to participate in bouton formation by releasing a synaptogenic cue, and to clear shed neuronal membrane which also impacts bouton formation. PG on the other hand have been hypothesized to respond to neuronal activity and match neuronal growth induced by chronic temperature raising (Tstim). Despite the efforts to characterize glial contributions to activity-dependent plasticity at the NMJ, some questions remain unanswered, such as can both SPG and PG sense activity at the NMJ? Does glia respond to activity with specific morphological changes? Are there specific types of glial protrusions involved in activity-dependent bouton formation? What mechanisms govern the formation these glial membranous structures? Do glial subtypes have redundant or synergetic roles at the NMJ? To address these questions, we subjected larvae with membrane labelled SPG and PG to acute and chronic activity protocols of stimulation. Our results are still preliminary but suggest that glial area does not change significantly after the induction of activity protocols to the NMJ. Nevertheless, both PG and SPG responded to Tstim with changes in the frequency of distinct membranous processes. Interestingly, we observed processes in very close contact with non-mature presynaptic ghost boutons but the relevance of this is still elusive. In unstimulated controls, we observed gliopods from both SPG and PG juxtaposed to ghost boutons. To further understand how glial subtypes interact between themselves, we did dual labelling of SPG and PG and observed processes of these glia do not always extend towards the same location, suggesting that they may be sensing different cues. We performed in vivo live imaging of labelled glia and observed that glial protrusions are dynamic and show differential calcium levels throughout the processes. Overall, our results suggest that SPG and PG adapt to chronically induced neuronal growth and activity and that possible local calcium dynamics exist within SPG processes after stimulation. Our findings also suggest non-redundant roles for SPG and PG considering their morphologies are distinct within single NMJs. Our results also suggest that gliopods from SPG and PG can modulate presynaptic structural plasticity given their close proximity with ghost boutons. Further understanding glial dynamics and intercellular communication at the NMJ will probably elucidate the mechanisms governing activity-dependent plasticity. |
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| Main Authors: | Pinho, Mariana Courteilles David |
| Subject: | Drosophila melanogaster |
| Year: | 2023 |
| Country: | Portugal |
| Document type: | master thesis |
| Access type: | embargoed access |
| Associated institution: | Universidade Nova de Lisboa |
| Language: | English |
| Origin: | Repositório Institucional da UNL |
| Summary: | Synaptic plasticity is a process by which neurons remodel their structure and/or molecular composition of synapses. This mechanism underlies nervous system connectivity and allows for cognition and memory formation. The Drosophila peripheral nervous system has been extensively used to characterize mechanisms of plasticity. The peripheral nerves are wrapped by three glial layers: the innermost wrapping glia (WG), middle layer of subperineurial glia (SPG) and the outermost perineurial glia (PG). The SPG and PG subtypes extend multiple membranous processes (namely lammelipodia-like structures, gliopods and gliobuls) into the larval neuromuscular junctions (NMJs). The NMJ synapse is established between motor neuron presynaptic terminals that innervate muscle cells and enables robust analysis of activity-induced structural plasticity. On motor terminals of 3rd instar NMJs, new boutons are added to the existing NMJ in response to developmental cues and in an activity-dependent manner. New boutons can be induced acutely through stimulation protocols based on patterned high K+ stimulation, or chronically by raising larvae at 290C, which increases larval locomotion and therefore activity. SPG at the NMJ has been previously shown to participate in bouton formation by releasing a synaptogenic cue, and to clear shed neuronal membrane which also impacts bouton formation. PG on the other hand have been hypothesized to respond to neuronal activity and match neuronal growth induced by chronic temperature raising (Tstim). Despite the efforts to characterize glial contributions to activity-dependent plasticity at the NMJ, some questions remain unanswered, such as can both SPG and PG sense activity at the NMJ? Does glia respond to activity with specific morphological changes? Are there specific types of glial protrusions involved in activity-dependent bouton formation? What mechanisms govern the formation these glial membranous structures? Do glial subtypes have redundant or synergetic roles at the NMJ? To address these questions, we subjected larvae with membrane labelled SPG and PG to acute and chronic activity protocols of stimulation. Our results are still preliminary but suggest that glial area does not change significantly after the induction of activity protocols to the NMJ. Nevertheless, both PG and SPG responded to Tstim with changes in the frequency of distinct membranous processes. Interestingly, we observed processes in very close contact with non-mature presynaptic ghost boutons but the relevance of this is still elusive. In unstimulated controls, we observed gliopods from both SPG and PG juxtaposed to ghost boutons. To further understand how glial subtypes interact between themselves, we did dual labelling of SPG and PG and observed processes of these glia do not always extend towards the same location, suggesting that they may be sensing different cues. We performed in vivo live imaging of labelled glia and observed that glial protrusions are dynamic and show differential calcium levels throughout the processes. Overall, our results suggest that SPG and PG adapt to chronically induced neuronal growth and activity and that possible local calcium dynamics exist within SPG processes after stimulation. Our findings also suggest non-redundant roles for SPG and PG considering their morphologies are distinct within single NMJs. Our results also suggest that gliopods from SPG and PG can modulate presynaptic structural plasticity given their close proximity with ghost boutons. Further understanding glial dynamics and intercellular communication at the NMJ will probably elucidate the mechanisms governing activity-dependent plasticity. |
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