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outgrowth. To evaluate the locust embryo as a test system for such chemicals, the accepted test compound rotenone, inhibiting axon elongation, as well as endpoint specific controls for cytoskeletal dynamics (colchicine and cytochalasine D) and calcium signaling (verapamil and diltiazem) were used. I focused on the effects of the calcium channel blockers and the effects of altered calcium levels on Ti1 pioneers.
Intracellular calcium concentrations change considerably throughout Ti1 pioneer neuron development. Using intracellular injection of calcium indicators, Bentley et al. (1991) could show that guidepost cells are contacted by Ti1 pioneers and cause a downregulation of intracellular calcium levels. Lau et al. (1999) have demonstrated that local increase of intracellular calcium in locust growth cones promotes the generation of filopodia. Intracellular calcium can be elevated either through the release of intracellular calcium stores or by the influx through ion channels (Berridge 1997, Walz et al. 1995, Lohr 2003). Continuous blockage of ion channels that allow calcium influx, such as voltage gated calcium channels, may lead to overall decreased intracellular calcium concentrations. Verapamil and diltiazem, two classes of L-Type calcium channel blockers that are also effective in insects (Lohr et al. 2005), inhibited Ti1 outgrowth in a concentration dependent manner, with neurite outgrowth being affected at lower concentrations than general viability. Since two different pharmacological classes of channel blockers were used, unspecific actions of both blockers on pioneer outgrowth seem to be unlikely. The gathered data implies that neurite elongation may be dependent on intracellular baseline calcium concentrations, given that blockage of calcium channels does indeed reduce calcium levels in the growth cone. Since localized calcium increase in the growth cone facilitates the formation of filopodia, reducing extracellular calcium influx may in turn reduce growth speed, as fewer filopodia are formed by the growth cone during navigation.
Intracellular calcium concentrations can be increased by the application of high concentrations of caffeine. This effect is caused by ryanodine receptor activation and subsequent release of intracellular calcium from the endoplasmic reticulum (Walz et al. 1995). Following the experimental protocol by Bode et al. (2020 b), dose response curves of caffeine were generated showing a concentration dependent disruption of correct pathfinding (Fig. 3). In contrast to experiments with calcium channel blockers, caffeine had no effects on axon elongation and general viability at tested concentrations (Fig. 3 A). However, pathfinding and fasciculation of
Isbister et al. (1999) where immunological block of Sema1 resulted in a similar phenotype.
Bentley et al. (1991) have shown that intracellular calcium concentrations are decreased in the Ti1 axon in the trochanter segment, while the growth cones travel along the band of Sema1 (Kolodkin et al. 1992). As there are currently no direct calcium imaging data of Ti1 growth cones available, it is unknown whether this decrease in calcium concentration in the axon is indicative for the calcium concentration in the growth cone. However, if this decrease extends
Fig. 3 Caffeine induces pathfinding errors in Ti1 pioneer neurons of the developing locust hindleg. Caffeine is known for its depleting effect on intracellular calcium stores at high concentrations. According to the experimental protocol described by Bode et al. (2020 b), I generated dose response curves (A) of two viability (resazurine and dead-cell protease assay), and two neurite growth endpoints (elongation score and correct pathways). n indicates the number of evaluated embryos. Viability and elongation were not affected at any tested concentration. However, caffeine induced pathfinding errors were observed (IC50: 20 mM).
Ti1 pioneer neurons develop stereotypically in embryo culture (B) as described in Bergmann et al. (2019). Incubation of developing embryos with caffeine disrupted correct neurite growth in a concentration dependent manner (C & D dented arrowheads). Note that increasing caffeine concentrations (C & D) affect the quantity of erroneous growth over all specimen and not the severity. Soma marked by asterisk, end of axon marked by filled arrowhead.
Scalebar = 100 µm.
decrease of intracellular calcium leads to reduced neurite growth, whereas calcium increase disrupts correct pathway formation in the trochanter segment. Interestingly, these effects are not in opposition to each other, suggesting that intracellular calcium concentrations in Ti1 growth cones has to be tightly regulated to facilitate proper steering and outgrowth. Whether this regulation is necessary for Sema1 guided growth in the trochanter segment remains a challenging question for future research.
To resolve pioneer neuron elongation and pathfinding errors in the three dimensional limb, scanning laser optical tomography was employed for imaging and metric assessment of neurite length. Whereas glycerol clears embryos sufficiently for epifluorescence microscopy, scanning laser optical tomography requires even less opacity of the tissue to reduce light scattering.
A mixture of methyl salicylate and benzyl benzoate has better tissue infiltration properties due to its low viscosity, reducing light scattering considerably. However, suspending tissue in clearing agents with low viscosity led to insufficient spatial fixation, resulting in movement artifacts during image acquisition. Ultimately, embryos were cleared using CRISTAL (Curing Resin-Infiltrated Sample for Transparent Analysis with Light), which utilizes polymerization of clearing agents under UV irradiation (Kellner et al. 2016), enabling simultaneous clearing and immobilization of the specimen. This allowed detection of arsenite induced erroneous outgrowth of Ti1 pioneer axons using the SLOT method. Thus, SLOT can be used as a rapid and automated imaging tool in future studies of erroneous outgrowth in DNT assays.
To confirm the effects of pharmacological agents for manipulation of calcium homeostasis directly, monitoring of cytosolic calcium is required. In organisms like Drosophila, introduction of genetically encoded calcium sensors allows for targeted imaging of specific neurons (Fiala et al. 2002). In a historical context, this advance is based on decades of research into Drosophila genetics (Brand and Perrimon 1993, Adams et al. 2000, Rubin 2000). The genome of Locusta migratoria has only been sequenced a few years ago (Wang et al. 2014) and a first draft of the Schistocerca gregaria genome has been published very recently (Verlinden et al. 2020). Considering the enormous size of locust genomes (6.5 Gb and 8.8 Gb respectively), which are 2 to 3 times larger than the human genome and over 50 to 70 times larger than the genome of Drosophila, it comes to no surprise that genetic manipulation of these animals is extremely challenging. As of yet, no genetic tools allowing manipulations on the scale necessary to achieve reliable simultaneous calcium imaging of defined locust neurons
indicators (Isaacson and Hedwig 2017). This severely limits the number of cells that can be monitored simultaneously for changes in their intracellular calcium levels. For imaging of large cell populations, calcium indicators have to be introduced by passive uptake over the cell membrane. This is commonly achieved by binding an acetoxymethyl ester (AM) group to calcium indicators, allowing for free diffusion over the membrane. Once inside the intracellular lumen, the indicator is trapped by hydrolysis of the AM group, which in turn allows calcium dependent fluorescence of the indicator. However, calcium indicators that are delivered in such a manner are quickly compartmentalized or extruded from locust neurons. I found that the calcium indicator Cal-520 AM readily loads locust neurons and is largely resistant to extrusion and compartmentalization, allowing for investigation of calcium dynamics.
Calcium indicators like Cal-520 AM can be utilized in future research for neurotoxicity assays.
The developmental neurotoxicity assay presented by our lab (Bode et al. 2020 b) has the disadvantage that it cannot differentiate between inhibited neurite growth due to DNT effects and neurotoxic effects. Since disruption of neurite outgrowth and cell death of the developing neurons cause the formation of a functioning nervous system to fail, both effects need to be differentiated as they are based on different molecular mechanisms. Understanding the specific molecular mechanisms that are disrupted by a DNT compound is crucial for treatment of developmental neurotoxic effects. Long term changes in intracellular calcium concentrations in response to chemical exposure are indicative of neurotoxic effects (Tymianski et al. 1993).
Due to its intracellular retention, Cal-520 AM can be used as a fluorometric sensor for calcium levels to differentiate general neurotoxicity from developmental neurotoxicity in cultured locust neurons.
Using the next generation calcium indicator Cal-520 AM, I aimed to obtain live calcium imaging data of pioneer neurons during development. In order to monitor intracellular calcium levels in response to pharmacological manipulation I tried to replicate filet preparations of locust metathoracic limb buds (Lefcort and Bentley 1987, Bentley et al. 1991) for subsequent loading with Cal-520 AM. However, in contrast to experiments with Schistocerca gregaria, embryonic tissue of Locusta migratoria did not sufficiently adhere to coated cover slips for stable recording.
as in isolated neuron culture of insects (Bicker and Kreissl 1994, Campusano et al. 2007, Oertner et al. 1999). Since Cal-520 AM readily loads locust neurons, I tried to elucidate neurophysiological and neuroanatomical aspects of neurons in the olfactory system of Locusta migratoria. Since the number of antennal lobe neurons as well as its respective subpopulations of projection neurons and local interneurons were unknown for Locusta migratoria, the number of cells within the antennal lobe was ascertained. Accounting for glia (Gocht et al. 2009), the antennal lobe is estimated to be comprised of about 1000 neurons. Calculations based on diameter distributions allowed me to estimate that roughly 24 % of antennal lobe neurons are local neurons. These numbers correspond nicely to data from the locust species Schistocerca gregaria, where the antennal lobe is comprised of 1130 neurons with 26 % local neurons (Laurent 1996, Leitch and Laurent 1996). Ernst et al. (1977) reported that soma diameters of antennal lobe neurons in the dorsal frontal cap measure between 20 µm and 25 µm, whereas somata that lie more ventrally range between 10 µm and 12.5 µm.
However, I found a more gradual distribution of soma diameters between 10 µm and 35 µm, making identification of local interneurons and projections neurons based on diameter difficult.
Receiver operating characteristic analysis (ROC) can be used for distinguishing individuals in a population, based on a suitable criterion. ROC calculates the probabilities of false negative and false positive results at a given criterion threshold, dividing a population into two subpopulations. Using ROC analysis, soma diameter was found to be a suitable criterion for the prediction of projection/local neuron identity and a threshold diameter of about 20 µm was defined for projection/local neuron classification.
Antennal lobe neurons respond to cholinergic stimulation with an increase of cytosolic calcium and also show a wide range of responsiveness towards nicotinic or muscarinic stimulations.
Surprisingly, muscarinic calcium responses in projection neurons were found whereas Drosophila projection neurons were reported to be unresponsive to muscarinic stimulations (Rozenfeld et al. 2019). This range of nicotinic and muscarinic responsiveness in projection neurons could indicate the presence of physiologically distinct subpopulations of projection neurons (Croset et al. 2018), expressing different types or quantities of nicotinic and muscarinic receptors. Local interneurons were observed to respond differently to cholinergic stimulation than projection neurons, possibly as a consequence of the different role they fulfill in the olfactory circuit. Particularly GABAergic local neurons were more responsive to muscarinic stimulation than other local neurons. GABAergic local neurons appear to be essential for the
responsiveness of local interneurons was detected after nicotinic/muscarinic co-stimulation, suggesting short term memory-like increase of cholinergic responsiveness. In Drosophila, a similar mechanism seems to be at play (Rozenfeld et al. 2019). There, muscarinic receptors play an important role in the mitigation of short term depression of GABAergic local neurons in the olfactory circuit. In locusts, this effect on nicotinic responsiveness was found in all local neuron types, but not in all individual local neurons.
The identification of a calcium indicator that readily loads and is retained by locust neurons opens up a new avenue of research in the development of locust specific pesticides.
Calcium imaging can be used to identify compounds that specifically disrupt locust neurophysiology in vivo and in culture. Moreover, recent advancements in genome editing with CRISPR-Cas9 rekindled research regarding the locust olfactory system (Li et al. 2016).
Elucidation of the locust olfactory network utilizing calcium imaging will provide further insight in the network mechanisms and cellular properties of the locust olfactory system that can be exploited for locust pest control. In conclusion, locusts remain highly relevant as devastating pest insects, as a test system for developmental neurotoxicity and as a fruitful preparation for basic neurobiology.
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