A microfluidics-based method for measuring neuronal activity in Drosophila chemosensory neurons

Abstract

Animals possess highly specialized sensory neurons that are capable of perceiving information about a continuously changing environment. Measuring neuronal activity in these cells is an important step toward understanding the basic coding principles, as well as the spatial and temporal dynamics, of sensory systems. The fruit fly Drosophila melanogaster is widely used as a genetic model organism to uncover basic mechanisms of taste coding and odor perception [1][2][3][4][5][6][7] . Recent insights into the molecular and functional mechanisms of sensory perception in Drosophila have been achieved by the use of single-sensillum recordings of adult taste or olfactory sensilla 1,4,[8][9][10][11][12][13] . This method enables sensitive measurements of action potential patterns and shows whether responses are excitatory or inhibitory, but drawing final conclusions about the individual firing neuron is difficult, as each sensillum contains two to four neurons 14 . In the larva, multicellular electrophysiological measurements of the taste system have been performed, but this method is technically challenging and it is not clear how many taste neurons are housed in which sensillum 15 .Microfluidic devices have been used previously to image command interneurons (AVA) and sensory neurons (ASH) in the nematode Caenorhabditis elegans and for studies of axotomy in peripheral sensory neurons of Drosophila larvae [16][17][18] . To implement a similar strategy for Drosophila larval chemosensation, we developed a simple and widely applicable microfluidic chip. We used this custom-made microfluidic device to deliver precise and temporally restricted tastant stimulation in the first published physiological characterization of individual larval gustatory receptor neurons (GRNs) expressing the calcium sensor GCaMP5 (ref. 19). Development of the protocolThe design of the chip is simple. It contains a single channel that is connected to a chamber in which the sample can be mounted to bring it into contact with fluids in the channel. The chamber was originally designed to exactly fit the head of a third-instar Drosophila larva; however, it is also suitable for the proboscis or the leg of an adult fly. The channel for delivering stimulants is relatively broad, designed at a width of 1 mm to reduce flow pressure on the sample. We use a single-channel system in which changes of liquids are performed by the tubing system outside the chip, allowing a relatively slow flow rate inside the chip. Here we provide information required for the design and production of this microfluidic device ( Fig. 1; Supplementary Data), as well as a detailed protocol for live imaging to acquire GCaMP-based calcium imaging (Figs. 2 and 3). By introducing the larval head, or the adult proboscis or leg, into the chamber, neuronal activity can be recorded while chemosensory neurons are in contact with the solution pumped through the channel (Figs. 2 and 3).Overview of the procedure An overview of the workflow involved in implementing the procedure is provided in Figu...

A receptor and neuron that activate a circuit limiting sucrose consumption

“…Once the stimulus perfuses through the system to the neuron and the Ca 2+ response becomes detectable, it takes on the order of 30 s to reach peak amplitude (Figure 7D,E). This rise time is comparable to that observed for some tastants in pharyngeal neurons of the larval taste system (Apostolopoulou et al, 2016; Choi et al, 2016; van Giesen et al, 2016b). However, the rise time of the Gr43a neuron, once it begins to respond to sugar, is faster (LeDue et al, 2015), suggesting that the Gr43a neuron activates a circuit that stimulates feeding before the IR60b neuron activates a circuit that inhibits feeding.…”

Collective behaviors of Drosophila-derived retinal progenitors in controlled microenvironments

“…Here, we leveraged the unique wealth of Drosophila data for direct comparison of total cell numbers and cell lineage over different stages of retinal development. We note that cells isolated from developing eye brain complexes of the third instar stage are neuroblasts, known to only differentiate into retinal neurons or glia during the later stages of development [28,62]. These RPCs have been shown to respond to stimuli collectively, in vivo, by a variety of studies using genetics with live imaging techniques [28,63] as well as conventional fixation over time [4,55,64].…”

Invertebrate Retinal Progenitors as Regenerative Models in a Microfluidic System

“…This invertebrate model, thereby, provides unique opportunities for development of biomaterials used in regenerative therapies for inherited diseases and retinal disorders more broadly [77]. In vitro study of the collective behaviors of Drosophila progenitors has been surprisingly scarce [52,78,79,80,81], largely because cells extracted from developing organisms are notoriously difficult to maintain in vitro: The average viability of cells isolated from Drosophila visual system has been reported to be 12% after 24 h [46,82]. We here report achieving cell viability approaching 80% after 48 h in vitro (Figure 5), presumably by using sterility protocols of mammalian cell culture in combination with specific substrate-coated surfaces (Figure 1).…”