Biology subjects

Aimino, M. A.

Publications and source records attributed to Aimino, M. A..

4 recordsLinked to original sources

bioRxiv2023

SynLight: a dicistronic strategy for simultaneous active zone and cell labeling in the Drosophila nervous system

At synapses, chemical neurotransmission mediates the exchange of information between neurons, leading to complex movement behaviors and stimulus processing. The immense number and variety of neurons within the nervous system makes discerning individual neuron populations difficult, necessitating the development of advanced neuronal labeling techniques. In Drosophila, Bruchpilot-Short and mCD8-GFP, which label presynaptic active zones and neuronal membranes, respectively, have been widely used to study synapse development and organization. This labeling is often achieved via expression of two independent constructs by a single binary expression system, but expression can weaken when multiple transgenes are expressed by a single driver. Ensuring adequate expression of each transgene is essential to enable more complex experiments; as such, work has sought to circumvent these drawbacks by developing methods that encode multiple proteins from a single transcript. Self-cleaving peptides, specifically 2A peptides, have emerged as effective sequences for accomplishing this task. We leveraged 2A ribosomal skipping peptides to engineer a construct that produces both Bruchpilot-Short and mCD8-GFP from the same mRNA, which we named SynLight. Using SynLight, we visualized the putative synaptic active zones and membranes of multiple classes of olfactory, visual, and motor neurons and observed correct separation of signal, confirming that both proteins are being generated separately. Furthermore, we demonstrate proof-of-principle by quantifying synaptic puncta number and neurite volume in olfactory neurons and finding no difference between the synapse densities of neurons expressing SynLight or neurons expressing both transgenes separately. At the neuromuscular junction, we determined that synaptic puncta number labeled by SynLight was comparable to endogenous puncta labeled by antibody staining. Overall, SynLight is a versatile tool for examining synapse density in any nervous system region of interest and allows new questions to be answered about synaptic development and organization.

neuroscience↗

bioRxiv2022

A conditional strategy for cell-type specific labeling of endogenous excitatory synapses in Drosophila reveals subsynaptic architecture

Chemical neurotransmission occurs at specialized contacts where presynaptic neurotransmitter release machinery apposes clusters of postsynaptic neurotransmitter receptors and signaling molecules. A complex program underlies recruitment of pre- and postsynaptic proteins to sites of neuronal connection and enables the correct three-dimensional synaptic organization that underlies circuit processing and computation. To better study the developmental events of synaptogenesis in individual neurons, we need cell-type specific strategies to visualize the individual proteins at their endogenous levels at synapses. Though such strategies exist for a variety of presynaptic proteins, postsynaptic proteins remain less studied due to a paucity of reagents that allow visualization of endogenous individual postsynapses in a cell-type specific manner. To study excitatory postsynapses, we engineered dlg1[4K], a conditional, epitope-tagged marker of the excitatory postsynaptic density in Drosophila. In combination with binary expression systems, dlg1[4K] effectively labels postsynaptic regions at both peripheral neuromuscular and central synapses in larvae and adults. Using dlg1[4K], we find distinct rules govern the postsynaptic organization of different adult neuron classes, that multiple binary expression systems can concurrently label pre- and postsynaptic regions of synapses in a cell-type-specific manner, and for the first time, visualize neuronal DLG1 at the neuromuscular junction. These results validate a novel strategy for conditional postsynaptic labeling without the caveats of overexpression and demonstrate new principles of subsynaptic organization. The use of dlg1[4K] marks a notable advancement in studying cell-type specific synaptic organization in Drosophila and the first example of a general postsynaptic marker to complement existing presynaptic strategies.

neuroscience↗

bioRxiv2022

Different olfactory neuron classes use distinct temporal and molecular programs to complete synaptic development

Developing neurons must meet core molecular, cellular, and temporal requirements to ensure the correct formation of synapses, resulting in functional circuits. However, because of the vast diversity in neuronal class and function, it is unclear whether or not all neurons use the same organizational mechanisms to form synaptic connections and achieve functional and morphological maturation. Moreover, it remains unknown if neurons united in a common goal and comprising the same sensory circuit develop on similar timescales and using identical molecular approaches to ensure the formation of the correct number of synapses. To begin to answer these questions, we took advantage of the Drosophila antennal lobe, a model olfactory circuit with remarkable genetic access and synapse-level resolution. Using tissue-specific genetic labeling of active zones, we performed a quantitative analysis of synapse formation in multiple classes of neurons throughout development and adulthood. We found that olfactory receptor neurons (ORNs), projection neurons (PNs), and local interneurons (LNs) each have unique time-courses of synaptic development, addition, and refinement, demonstrating that each class follows a distinct developmental program. This raised the possibility that these classes may also have distinct molecular requirements for synapse formation. We genetically altered neuronal activity in each neuronal subtype and observed differing effects on synapse number based on the neuronal class examined. Silencing neuronal activity in ORNs, PNs, and LNs impaired synaptic development but only in ORNs did enhancing neuronal activity influence synapse formation. ORNs and LNs demonstrated similar impairment of synaptic development with enhanced activity of a master kinase, GSK-3{beta}, suggesting that neuronal activity and GSK-3{beta} kinase activity function in a common pathway. ORNs also, however, demonstrated impaired synaptic development with GSK-3{beta} loss-of-function, suggesting additional activity-independent roles in development. Ultimately, our results suggest that the requirements for synaptic development are not uniform across all neuronal classes with considerable diversity existing in both their developmental timeframes and molecular requirements. These findings provide novel insights into the mechanisms of synaptic development and lay the foundation for future work determining their underlying etiologies.

neuroscience↗

bioRxiv2018

The Tenets of Teneurin: Conserved Mechanisms Regulate Diverse Developmental Processes in the Drosophila Nervous System

To successfully integrate a neuron into a circuit, a myriad of developmental events must occur correctly and in the correct order. Neurons must be born and grow out towards a destination, responding to guidance cues to direct their path. Once arrived, each neuron must segregate to the correct sub-region before sorting through a milieu of incorrect partners to identify the correct partner with which they can connect. Finally, the neuron must make a synaptic connection with their correct partner; a connection that needs to be broadly maintained throughout the life of the animal while remaining responsive to modes of plasticity and pruning. Though many intricate molecular mechanisms have been discovered to regulate each step, recent work showed that a single family of proteins, the Teneurins, regulates a host of these developmental steps in Drosophila - an example of biological adaptive reuse. Teneurins first influence axon guidance during early development. Once neurons arrive in their target regions, Teneurins enable partner matching and synapse formation in both the central and peripheral nervous systems. Despite these diverse processes and systems, the Teneurins use conserved mechanisms to achieve these goals, as defined by two tenets: 1) transsynaptic interactions with each other and 2) membrane stabilization via an interaction and regulation of the cytoskeleton. These processes are further distinguished by 1) the nature of the transsynaptic interaction - homophilic interactions (between the same Teneurins) to engage partner matching and heterophilic interactions (between different Teneurins) to enable synaptic connectivity and the proper apposition of pre- and postsynaptic sites and 2) the location of cytoskeletal regulation (presynaptic cytoskeletal regulation in the CNS and postsynaptic regulation of the cytoskeleton at the NMJ). Thus, both the roles and the mechanisms governing them are conserved across processes and synapses. In this review, we will highlight the contributions of Drosophila synaptic biology to our understanding of the Teneurins and discuss the mechanistic conservation that allows the Teneurins to achieve common neurodevelopmental goals. Finally, we will posit the next steps for understanding how this remarkably versatile family of proteins functions to control multiple distinct events in the creation of a nervous system.

neuroscience↗