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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Neurochem Int. 2020 Apr 18;137:104744. doi: 10.1016/j.neuint.2020.104744

Deficits in the vesicular acetylcholine transporter alter lifespan and behavior in adult Drosophila melanogaster

Daniel White 1,*, Raquel P de Sousa Abreu 2,*, Andrew Blake 1, Jeremy Murphy 1, Shardae Showell 1, Toshihiro Kitamoto 3, Hakeem O Lawal 1
PMCID: PMC7247942  NIHMSID: NIHMS1585811  PMID: 32315665

Abstract

The neurotransmitter acetylcholine (ACh) is involved in critical organismal functions that include locomotion and cognition. Importantly, alterations in the cholinergic system are a key underlying factor in cognitive defects associated with aging. One essential component of cholinergic synaptic transmission is the vesicular ACh transporter (VAChT), which regulates the packaging of ACh into synaptic vesicles for extracellular release. Mutations that cause a reduction in either protein level or activity lead to diminished locomotion ability whereas complete loss of function of VAChT is lethal. While much is known about the function of VAChT, the direct role of altered ACh release and its association with either an impairment or an enhancement of cognitive function are still not fully understood. We hypothesize that point mutations in Vacht cause age-related deficits in cholinergic-mediated behaviors such as locomotion, and learning and memory. Using Drosophila melanogaster as a model system, we have studied several mutations within Vacht and observed their effect on survivability and locomotive behavior. Here we report for the first time a weak hypomorphic Vacht allele that shows a differential effect on ACh-linked behaviors. We also demonstrate that partially rescued Vacht point mutations cause an allele-dependent deficit in lifespan and defects in locomotion ability. Moreover, using a thorough data analytics strategy to identify exploratory behavioral patterns, we introduce new paradigms for measuring locomotion-related activities that could not be revealed or detected by a simple measure of the average speed alone. Together, our data indicate a role for VAChT in the maintenance of longevity and locomotion abilities in Drosophila and we provide additional measurements of locomotion that can be useful in determining subtle changes in Vacht function on locomotion-related behaviors.

Keywords: vesicular acetylcholine transporter, cholinergic release, acetylcholine

INTRODUCTION

Aging is an inevitable physiological process that is shared across all animal species and involves a decline in essential physiological functions (He and Jasper, 2014). The biology of aging is complex and is thought to be characterized in part by the generation of free radicals which are a byproduct of metabolism (Harman, 1981; He and Jasper, 2014). Ultimately, aging affects cells, tissues, and organs adversely and, not surprisingly, it is a risk factor for diseases such as Alzheimer’s disease, cancer, cardiovascular disease, and diabetes (DiLoreto and Murphy, 2015; Wahl et al., 2019). The exact causes of aging remain unknown, but a common theory is that it results from the progressive accumulation of cellular, DNA, and protein damage due to risk factors such as oxidative stress (He and Jasper, 2014; Liochev, 2015; Zimniak, 2012).

The contribution of neurotransmitters to aging has been investigated in detail (Chen et al., 2005; Dickinson-Anson et al., 2003; Neckameyer et al., 2000; Schliebs and Arendt, 2011; Sugita et al., 2016; Thiruchelvam et al., 2003; Turrini et al., 2001). ACh in particular, has been proposed to play a key role in the physiological decline associated with aging (Dickinson-Anson et al., 2003; Schliebs and Arendt, 2011; Sugita et al., 2016; Turrini et al., 2001). ACh is synthesized in the cytoplasm of cholinergic neurons by the enzyme choline acetyltransferase using acetyl coenzyme A and choline (Greenspan et al., 1980; Varoqui and Erickson, 1996). After its synthesis, ACh is packaged into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Interestingly, aging is characterized in part by a decline in the expression of cholinergic neuronal proteins (Schliebs and Arendt, 2011). Moreover, Alzheimer’s disease, which can be described as a case of pathological aging, is characterized in part by the death of cholinergic neurons (Ferreira-Vieira et al., 2016; Lombardo and Maskos, 2015). The evidence of direct causality between aging and cholinergic transmission, however, remains inconclusive.

Because of its central role in ACh neurotransmission, VAChT is an important tool for manipulating ACh regulation. Indeed, previous reports in rodents have shown that VAChT overexpression causes an increase in ACh release and enhances aging in neuromuscular junctions (Kolisnyk et al., 2013; Sugita et al., 2016). Work in Drosophila has also helped elucidate a a role for VAChT in synaptic transmission. Fly VAChT was described two decades ago (Kitamoto et al., 2000) and shown to have a high degree of similarity to its mammalian cognate, including in its role in the regulation of synaptic physiology and locomotion. Research in Drosophila VAChT identified two impotant point mutations which lead to either a partial or a complete loss of protein function. Moreover, overexpression of Drosophila VAChT, like its mammalian homolog, causes severe deficits in learning and memory (Showell et al., 2020). Importantly, an overexpression of VAChT in Drosophila causes an age-dependent defect in locomotion (Showell et al., 2020). Notwithstanding these important findings, the importance of reduced VAChT function, and consequently synaptic ACh release, in the regulation of behavioral and cognitive processes during aging is still not fully understood.

One major component in uncovering insights into the mechanism of aging has been the use of behavioral assays to measure the state of neurological activity. Typically, studies in rodent and fly models have relied on locomotion activity as a readout for neurological performance and its changes during aging (Guzman et al., 2011; Kitamoto et al., 2000; Prado et al., 2006). Accordingly, different assays including average speed (Grygoruk et al., 2014; Pizzo et al., 2013), climbing assays (Chaudhuri et al., 2007; Coulom and Birman, 2004; Lawal et al., 2010), startle reflex (Lebestky et al., 2009), have been used to measure the contribution of diverse neurotransmitter systems. While the currently available measurements provided by these paradigms have helped uncover important roles for synaptic transmission in the regulation of those behaviors, they can be further explored and improved upon to better understand how neurons control behavior during the lifespan. To determine the effect of alterations in Vacht function on behavior during aging, we developed several new analytical tools for examining locomotion-related parameters that allowed us to measure and visualize aspects of locomotion that have not been previously explored in detail.

We tested the effect of reduced Vacht function on locomotion ability across the lifespan. We report that the Vacht mutant constructs we studied displayed differential defects in our measurements of locomotion that were, in general, dependent on the severity of the mutation. However, in a measurement for migration pattern of Drosophila in an open field, the effect of a loss in Vacht functionality was the similar without regard for the severity of the mutation. Strikingly, overexpression of VAChT produced a similar effect on migration suggesting the requirement of a defined level of VAChT expression for normal migratory behavior. These data demonstrate that ACh release may be involved in the regulation of the ability of Drosophila to display a stereotypical pattern of locomotion. Taken together, this work highlights an as-yet underappreciated role for ACh neurotransmission in the regulation of behavioral performance during the lifespan and suggests new ways of measuring the role of ACh signaling in mediating behavioral performance.

MATERIALS AND METHODS

Drosophila strains and culture conditions

Fly stocks w1118CS15 (w1118 outcrossed fifteen times to Canton-S), w; Chat-Gal4, UAS-GFP (BDSC #6793, referred to as Chat-Gal4 in the text and Figure 6), w; +; UAS-VAChT (Showell et al., 2020), w;P[Vacht]; Df (3R)Cha5/TM6B-ubi-GFP (a Vacht rescue construct containing P[Vacht]), a Vacht minigene, with a 7.4 kb genomic fragment covering the cholinergic locus fused with VAChT cDNA in the background of a 3rd chromosome deletion spanning the cholinergic locus (Kitamoto et al., 2000), Vacht1/TM6B-ubi-GFP, Vacht2/TM6B-ubi-GFP, Vacht8/TM6B-ubi-GFP, and the balancer stock w; Bl/CyO; TM2/TM6B-ubi, GFP were raised in a 12-hour light/dark cycle at 25°C and relative humidity of 50% on a sugar-yeast-corn meal diet which was produced in-house and poured into clean plastic vials. To prevent food from becoming either too soft or too dry, food vials were swapped for fresh ones every six days.

Figure 6. Overexpression of VAChT alters Drosophila exploratory behavior in an open field.

Figure 6.

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Young (3–7 day old) VAChT overexpressing flies showed a greater preference for the periphery of the assay arena compared with the center when we tested for their locomotion pattern in an open field. (A) Cha-Gal4/+ and UAS-VAChT/+ controls each showed a generally even distribution in the density of migration between the periphery and the center with a slight preference for the periphery of the arena. By contrast, locomotion in Cha-Gal4/UAS-VAChT expressing flies was concentrated along the edges of the arena with relatively sparse distribution around the center of the plate. Cha-Gal4/UAS-VAChT showed a significantly lower ratio (ANOVA with Tukey post-hoc test, n.s. p≥0.05; ** p≤10−5.) of center/total distribution compared to each of the controls. (B) Boxplot showing the quantification of the ratio C/(C+P), which indicates flies explored only the periphery (P) of the arena if equal to zero or that flies explored only the center (C) of the arena if equal to 1. The data are a representation of three independent experiments; N=9 per group.

Generation of partially rescued Vacht mutants

Vacht point mutations were generated by T. Kitamoto using ethylmethane sulfonate (EMS) as a mutagen (see (Kitamoto et al., 2000)). Vacht1 and Vacht2 are a putative null and a strong hypomorphic mutant allele, respectively (Kitamoto et al., 2000). Vacht8 is a previously unreported missense mutation resulting in a single amino acid substitution at position 345 (C345Y) in the transmembrane domain IX of the transporter (manuscript in preparation). In this study, w; +; Vacht/TM6B-GFP virgins were crossed to P[Vacht]/ P[Vacht]; Df(3R)Cha5/TM6B-GFP males to generate the Vacht partial rescue flies P[Vacht]/ +; Df(3R)Cha5/Vachtmutant (from here on referred to as Vachtmutant partial rescue or Vacht1-Res, Vacht2-Res and Vacht8-Res). We used the small deletion construct spanning the chromosome region (91B3;91D1) which includes the entire Vacht locus Df (3R)Cha5 to generate the Vacht [mutant]/D(f3R) Cha5 line in order to eliminate as much Vacht gene function as possible and to rule out any residual background genetic effect that may be associated with the generation of the point mutation via EMS mutagenesis. We collected the Vacht partial rescue flies using GFP as a negative selection under a Nikon SMZ18 stereomicroscope (Melville, NY, USA). The progeny were thus collected as non-GFP 2nd instar larvae and incubated until they reached adulthood.

Lifespan Assay

For each genotype, adult male Vacht partial rescue flies were collected and placed in cohorts such that the individuals were 2–9 days old. The total number of flies in each cohort was observed and recorded. The last day of the 7-day lifespan became day 0 of the lifespan assay. Every three days from Day 0, the total number of flies that died was recorded and subtracted from the total number of flies that were alive when the cohorts were established. This assay continued until all the flies were dead. To prevent flies from dying prematurely due to bad food, the cohorts were independently swapped into new vials containing food twice a week. Three independent experiments were perform, N=6–19 per group.

Locomotion Behavior Assay

To examine the locomotion behavior of the partially rescued Vacht mutants, we used the automated Multiworm Tracker (MWT) that was optimized for measuring locomotion in adult Drosophila (Showell et al., 2020). This real-time tracking system allows for rapid quantification of locomotive behavior of C. elegans (Swierczek et al., 2011) and Drosophila (Grygoruk et al., 2014; Pizzo et al., 2013) larvae with minimal human effort. Briefly, this system comprises a real-time image analysis software, the MWT, which converts images captured by an overhead camera Dalsa PT-41–04M60 (Toronto, CN) into pixels, which are then quantified and analyzed. Because MWT was developed for measuring behavior in C. elegans, it is unable to record movement in a three-dimensional field. We therefore optimized the system for measuring locomotion in adult Drosophila. First, we prevented the flies from jumping or flying by creating a two-dimensional arena. We made a film of Sylgard 170 silicone elastomer base (Dow Corning, Midland, MI) and placed it into both the petri dish and its cover, with the amount of Sylgard leaving just enough space for the flies to walk freely but unable to jump or fly. To introduce flies to the arena, we temporarily immobilized flies by placing them on ice for 5 seconds. We then quickly transferred the animals into the Sylgard-coated arena. We also made some important modifications to the detection parameters of the MWT such that it is able to detect adult Drosophila, which are significantly larger than the larvae. We used MWT to capture and record three to five individuals during a 90-second window. To allow the flies to acclimate to the environment and slow down their movement due to agitation and stress, the first 30 seconds of recording was omitted from the analyses. We then performed this assay once a week for the duration of the lifespan study. We chose the mornings to run all of the locomotion assays (except stated otherwise) in order to minimize any time-of-day variation in behavior.

Statistics and Data Analysis

Survival curves were analyzed using Prism software (GraphPad, San Diego, CA) and Microsoft Excel. The Log-rank (Mante-Cox) statistical analysis was performed on each WT vs. partial Vacht-Res pair.

Locomotion data was imported, organized, and analyzed in R: The R Project for Statistical Computing (R Foundation for Statistical Computing, Vienna, Austria). All plots and statistical tests were implemented using a custom R code. To determine statistical significance of changes, we used ANOVA and post-hoc Tukey tests for multiple comparisons. The analysis of repeated-measures of speed during the 60 seconds of the assay was done using the nparLD procedure developed previously (Noguchi K, 2012) to compute the relative treatment effect (RTE) and ANOVA-type statistic (ATS) (Erceg-Hurn and Mirosevich, 2008), which accurately controls Type I error rate. According to authors’ terminology (Noguchi K, 2012), we used the LD-F1 model, i.e., seconds as a within-subject factor (“sub-plot” repeated factors). For each week, Wild-type (WT) and each mutant were tested.

For the study of the relationship between speed and time (seconds), we implemented a Kendall–Theil Sen Siegel non-parametric linear regression model for WT and each partially rescued Vacht mutant each week. Estimates of the slope and Y intersect were computed for each regression model. Boxplots display the minimum, first quartile, median, mean, third quartile, and maximum. The box is drawn from the first quartile to the third quartile and represents the interquartile range (IQR), a measure of statistical dispersion of the data. Outliers are observations that are numerically distinct from the rest of the data, i.e., that are located 1.5 times the IQR above the upper quartile and bellow the lower quartile. Density plots were computed and drawn using a kernel density estimate of the histogram of the speeds.

RESULTS

Our experimental goal was to determine the effect of changes in ACh neurotransmission on behavioral performance during aging. To assess this effect, we measured lifespan and locomotion. Our first challenge was to generate adult animals with deficits in Vacht. Since Vacht is essential for survival, its loss is lethal (Kitamoto et al., 2000; Prado et al., 2006; Zhu et al., 2001). Therefore, we deployed some genetic manipulations to bypass the developmental lethality that results from the absence of that protein’s function. By expressing a wild type (WT) copy of Vacht driven by a 7.4 kb upstream regulatory sequence (see MATERIALS AND METHODS), we generated a construct with WT Vacht expressed in each of the mutant lines we studied, resulting in a partial loss of Vacht function, described in Kitamoto et al. (Kitamoto et al., 2000). We then used the partially rescued Vacht mutants to determine the role of decreased cholinergic neurotransmission on lifespan. We studied three independent point mutations which were recovered in a previous screen. Vacht1 and Vacht2 have been reported and described previously (Kitamoto et al., 2000). Vacht8 is a previously unreported mutant allele that carries a missense mutation resulting ina a cystein-to-tyrosine substitution at position 345 (see MATERIALS AND METHODS). The partially rescued Vacht mutants are also referred to in this manuscript as Vacht1-Res, Vacht2-Res, and Vacht8-Res, respectively. Importantly, we measured the effect of these mutations on locomotion-based assays using analytical tools that we developed for that purpose.

Survival

To assess the effect of a decrease in VAChT function on longevity, we tested for survivorship in the adult Vachtmutant partial rescue that we generated (see MATERIALS AND METHODS). We then scored survivorship in these flies throughout the lifespan. Fly cohorts consisted of flies that hatched within a week from each other and were no older than 9 days. We report that w1118CS15 control flies had an average, median and maximum survival duration of 63, 46 and 67 days respectively (Figure 1). By comparison, Vacht1-Res had an average, median and maximum survival duration of 33, 25 and 46 days respectively (statistical analysis: Log-rank test comparison to WT, p<0.0001). The hypomorph Vacht2-Res mutant (Kitamoto et al., 2000) had an average, median and maximum survival duration of 57, 25, 43 days (statistical analysis: Log-rank test comparison to WT, p<0.0001). Vacht8-Res mutant survived at an average, median and maximum survival duration of 62, 43 and 61 days respectively (statistical analysis: Log-rank test comparison to WT was not significantly different from median values of WT). Overall, the data suggest that deficits in the VAChT leads to a negative effect on longevity in flies but this severity varies with the type of mutation in Vacht, i.e., putative null Vacht1 or strong hypomorph Vacht2; vs. weak hypomorph, Vacht8.

Figure 1. Effects of allelic series of Vacht mutations on lifespan.

Figure 1.

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Partially rescued Vacht adult mutants show a differential effect on survivorship. Vacht1-Res shows the strongest phenotype showing a severe reduction in lifespan. Similarly, Vacht2-Res shows a strong decrease in survival. Vacht8-Res, by contrast, is indistinguishable from WT. The data are a representation of at least five independent experiments; N≥3 per experiment.

Analysis of average speed during the lifespan

As a first step towards analyzing the effect of a reduction in Vacht expression on behavior, we followed the average speed protocol that has been reported by others (Grygoruk et al., 2014; Pizzo et al., 2013). We simultaneously tracked the movement of up to n=5 flies within a cohort in a given week for four weeks. We then investigated differences on average speed (Figure 2; ANOVA and post-hoc Tukey test, n.s. p≥0.05, * 10−5<p<0.05, ** p≤10−5) among genotypes and across lifespan. Consistent with published reports (Simon et al., 2006), we observed that in WT, the average speed tended to decline within the first few weeks of life (wk0≈0.3 cm/s vs. wk4≈0.2 cm/s, p≤10−5). We observed the lowest average speed, three weeks after Day 0. Vacht2-Res showed a generally lower average speed but a similar trend to WT (wk0≈0.2 cm/s vs. wk4≈0.1 cm/s). By contrast, Vacht1-Res, which started with severely reduced average speed, ≈0.12cm/s, maintained a poor locomotion state throughout the period of the lifespan we studied without significant differences after the first week. Unlike WT or Vacht1-Res or Vacht2-Res, Vacht8-Res showed a significant increase during the first three weeks (wk0≈0.2 cm/s vs. wk3≈0.4 cm/s, p≤10−5) before showing a decline in week 4, wk4≈0.2 cm/s.

Figure 2. Effect of a Vacht mutation series on locomotion during aging.

Figure 2.

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The average locomotion speed (mean ± SEM, cm/s) was measured for three adult Vacht partial rescues across a 4-week period. In WT, significant changes were observed between consecutive weeks such that, at the end of wk4, average speed had dropped considerably (**) in comparison with wk0. By contrast, Vacht1-Res mutants showed no change in locomotion rate (n.s.) at any time throughout the 4-week period. Vacht2-Res showed a pattern similar to WT (*). The Vacht8-Res demonstrated an increase in locomotion until wk3 that is atypical of the decline in locomotion observed in WT under normal conditions. At wk4, however, there was a significant reduction in locomotion that was comparable to wk0 (n.s.). Moreover, each week the average speed in WT is also significantly distinct from each Vacht partial rescue strain (* or **). Statistical analyses were performed using ANOVA and post-hoc Tukey multiple comparisons. n.s. p≥0.05; *10−5<p<0.05; ** p≤10−5. The data are a representation of four independent experiments; N≥4 per group.

Analysis of speed over a short-term timescale (seconds)

To get a fuller understanding of the effect of changes in Vacht on locomotion behavior, we performed a more thorough and descriptive analysis of speed using individual time points (per second) during the assay. We used a robust rank-based method for the analysis of repeated-measures of speed (nparLD, a procedure developed previously Noguchi et al. (Noguchi K, 2012) in WT and Vacht partial rescue flies for each week. We found no significant changes on the speed of the WT flies nor the Vacht partial rescues throughout the duration of our locomotion assay, indicating that the speed is consistent at this short-term timescale (seconds) and that average speed is a suitable and representative measurement of locomotion behavior.

Assessing only the average speed may also conceal trends occurring over the time of the assay (1 minute). Throughout the 1 minute assay, flies might manifest, for instance, incremental or irregular changes on speed. Alternatively, speed might be maintained relatively constant. For more clarity concerning these potential trends in speed over time, we performed regression analyses to examine the relationship between speed and time (Figure 3). For the WT and partially rescued Vacht mutants we studied, we found consistent speed during the assay, as indicated by the slopes, which were found always around zero. No evident trends on the speed were therefore identified for either the WT or the partially rescued Vacht mutants. In these cases, the Y intersect provides a rough estimate of the average speed during the assay. We found that Vacht1-Res mutants are distinct from the other mutants as well as from WT, with consistently slower speed profile each week and over the lifespan we investigated.

Figure 3. Speed-Time plot analysis of Vacht mutants during aging reveals a uniform but differential effect among partially rescued Vacht mutant alleles.

Figure 3.

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Three partially rescued Vacht mutants alleles were analyzed for rate of locomotion at distinct times during the one-minute assay period across a five week period (week 0 at the top of the panel)s. Particularly in WT, speed-time plots were found more scattered. Nevertheless, WT and mutants displayed a consistent and generally uniform distribution of speed throughout the assay and lifespan, as indicated by the slope values from the regression models, which were estimated around zero. In these cases, the Y intersect values from the models provide a rough estimate of the average speed. High intersect estimates indicate high average speeds during the assay, e.g., Vacht8-Res in weeks 2 and 3. In contrast, low Y intersect estimates indicate low average speeds during the assay, e.g., Vacht1-Res in weeks 0 to 2. n.s. p≥0.05, *10−5<p<0.05; ** if p≤10−5. The data are a representation of four independent experiments; N≥4 per group.

Additional measurements of locomotion activity

We tested the flies during the first week (wk0) in order to investigate effects likely related to the specific mutant without any potential complication from age-related effects, we compared baseline locomotion behavior of WT during wk0 with the behavior of the other partial rescues. Using boxplots and density plots (Figure 4A; ANOVA and post-hoc Tukey test, n.s. p≥0.05, * 10−5<p<0.05, ** p≤10−5), we found that in wk0, the speed of WT was more dispersed (larger interquartile range, IQR) and with a broader density distribution. In contrast, speed in Vacht partial rescue flies was less dispersed (smaller IQR) and with a skewed density distribution toward lower speeds. Interestingly, Vacht partial rescue flies exhibited a higher percentage of outliers, i.e., speed values that deviated considerably from the other speeds. Particularly in Vacht8-Res, we found speeds significantly higher in 16% of the dataset. Showing that, at this early stage of the lifespan, this mutation is already impacting the speed toward higher values. The emergence of this distinct but underrepresented cluster was also identified as an additional density peak on the density plots. We report that averaged speed by itself does not detect these fine effects on locomotion, as it only captures the mean speed over the entire duration of the assay. We also tested weeks 1–4 to examine whether there were any effects of aging on the locomotion density and we found that in contrast to WT, Vacht1-Res consistently showed a high density of locomotion near 0 cm/sec with the exception being wk4 which showed a sharp drop in density at the same reduced speed. At the other end of the spectrum was Vacht8-Res which started out with a bimodal peak with a high density at low speed and a small peak with outliers at high speed. Over the first three weeks, however, the speed distribution of Vacht8-Res becomes broader toward higher speed values.

Figure 4. Box and density plots comparisons among WT and partially rescued Vacht mutants across time points during aging.

Figure 4.

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(A, top) Boxplots of WT, Vacht1-Res, Vacht2-Res, and Vacht8-Res reinforce differences in locomotion speed profiles. Vacht1-Res, the most severe allele, shows a consistent and more uniform low speed profile regardless of age while Vacht8-Res shows a more variable profile; increased average speed until week 4 then a decrease is observed. (A, bottom) Density plots of WT and all partially rescued Vacht mutants are compared each week throughout the duration of the study. Skewed distributions indicate speed profiles that are more uniform while wider distributions indicate more dispersion. Additionally, high points, or peaks, on the density plots represent speeds where data is more concentrated. A bimodal distribution has two peaks instead of one. Density plots complements observations in A. At week 0 both Vacht1 and Vacht8 partial rescues have a high density of flies at low speed compared to WT. Vacht8-res, however, shows an additional underrepresented, but distinct, peak at a higher speed. By weeks 1 and 2, a clear distinction has emerged between Vacht1-Res on one hand and Vacht2-Res, Vacht8-Res, and WT on the other hand. Notably at week 2, the shape of the density curve of Vacht2-Res and WT are quite similar; Vacht8-Res by contrast is distinct exhibiting both a shift in the peak toward a higher speed and a broader area representing a wide range of locomotion speed. At week 3, with the exception of Vacht8-Res, all density plots are skewed toward low speeds. At week 4, however, the density profile of Vacht8-Res becomes identical to WT and other partially rescued Vacht mutants. (B) Boxplots showing the Center/Center+Periphery for each genotype for wk0 through wk4. Statistical analyses were performed using ANOVA and post-hoc Tukey multiple comparisons. n.s. p≥0.05; *10−5<p<0.05; ** p≤10−5. The data are a representation of four independent experiments; N≥4 per group.

Taken together, our data indicate that while average speed is a valuable measurement to the identification of major effects on locomotion, it can potentially conceal other relevant features, like speed trends during the assay period.

Exploration in an open field

In each of the three assays we studied so far, we analyzed locomotion either by measuring average speed or through a characterization of individual speed data points. Here, we sought to determine whether decreased Vacht function had any effect on the pattern of the migratory behavior of flies in an open field. In rodents, animals show a preference for migrating at the periphery as a display of anxiety (Higaki et al., 2018; Masini et al., 2018; Pervolaraki et al., 2019). Drosophila also displays behaviors in which adults show a preference to the edge of the arena over the central zone (Besson and Martin, 2005; Hughson et al., 2018; Soibam et al., 2012), although it is not clear whether this is centrophobism (avoidance of the center) or thigmotaxis (a preference for movement around the periphery) (Soibam et al., 2012). Despite the fact that this behavior has been previously reported in Drosophila, the neurological circuit that regulates it have not been fully elucidated. Therefore, we reasoned that this type of assay could help uncover insights into the role of neurotransmitters like ACh in regulating navigation ability as opposed to locomotion rates alone. Using the data collected by the MWT, we traced the displacement of each fly throughout the assay period and recorded their movement. To quantify locomotion patterns in a circular chamber, we measured locations explored in the center of the arena and normalized by all locations explored, i.e., in the center (C) and periphery (P). A ratio C/(C+P)=0 indicates that flies explored only the periphery of the arena and a ratio C/(C+P)=1 indicates that flies explored only the center of the arena. WT flies localized in an apparently random pattern that was generally evenly distributed throughout the assay plate but with a slight preference for the edges (Figure 5). Moreover, this distribution persisted throughout the period of the lifespan measured. By contrast, we report that all three partially rescued Vacht mutants, were localized in a circular, ring-like pattern at the periphery of the assay plate and less so at the center. A behavior that was maintained throughout the period of the lifespan measured (Figure 5). Despite Vacht1-Res being the most severe of the mutations in this study (it is an embryonic lethal), it did not show a dramatic difference in exploratory pattern to Vacht2-Res (a strong hypomorph). Interestingly, the least severe mutation Vacht8 also displayed a similar location pattern to both Vacht1-Res and Vacht2-Res. While the peripheral location preference was maintained generally throughout the lifespan, in Vacht8-Res, there was relatively less of a preference for the periphery in the initial couple of weeks, but the peripheral preference effect was fully evident by the 4th week. In order to quantify the phenomena that we observed, we measured the number of locations explored at the center (inner circle) or periphery (outer circle) of the assay plate relative to all locations explored within the entire assay plate (Figure 4B; ANOVA and post-hoc Tukey test, n.s. p≥0.05, * 10−5<p<0.05, ** p≤10−5 and Figure 5) and we found that with the exception of Vacht1-Res at week 4, there was a statistically significant difference between WT and each of the partially rescued Vacht mutants at each week tested. We note that despite the similarity among the phenotypes observed, there is still some variability within the different mutant groups. For example, at week 0, Vacht2-Res appears to show a stronger shift to the periphery compared to other groups, whereas at week 2 and week 4 Vacht8-Res showed a more decision shift to the periphery.

Figure 5. Mutations in Vacht exacerbate a centrophobism or thigmotaxis-like behavior.

Figure 5.

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WT flies display a generalized exploratory behavioral pattern that shows a slight preference for the periphery of the arena when organisms are placed in a circular arena. This behavior is maintained through the duration of the lifespan studied. By contrast, all partially rescued Vacht mutants display a centrophobism (avoidance of the center) or thigmotaxis (a preference for movement around the periphery) -like behavior. Importantly, this phenotype does not appear dependent on the type or severity of the Vacht mutant allele. For visualization of this thigmotaxis-like behavior we used a color gradient to indicate areas of lower (blue) and higher (red) density of the explored locations. For its quantification, we measured the ratio C/(C+P), which indicates flies explored only the periphery (P) of the arena if equal to zero or that flies explored only the center (C) of the arena if equal to 1. Density plots of WT and mutants are shown for each week. The data are a representation of four independent experiments; N≥4 per group.

We were intrigued by the robustness of the migration pattern deficits that we observed in the partially rescued Vacht mutants and the fact that the effect occurred regardless of the severity of the mutation. We reasoned that this behavioral circuit, unlike the baseline locomotion, is sensitive to subtle deficits in ACh levels. To test this idea, we examined locomotion speed and patterns in flies that overexpress Vacht. We had observed that baseline locomotion rates were not affected in these lines at 7 days of age (Showell et al., 2020). We therefore tested the flies at that stage of their lifespan and asked whether Vacht function affects locomotion patterns in a circular arena. We report that the Vacht overexpressor showed a strong and statistically significant effect (Figure 6) in which those flies were distributed toward the periphery relative to the center compared to Cha-Gal4/+ and UAS-VAChT/+. Moreover, while both control groups appear to show slightly greater distribution of migration along the edges, the effects in the control are relatively mild compared to the Vacht overexpressing flies.

Together, these data show that Vacht partial rescues display a similar behavioral pattern in the migration assay regardless of the severity of the mutation, in contrast to the measurements of baseline locomotion which show allelic specific differences in movement.

DISCUSSION

The precise role that cholinergic neurotransmission plays in the decline of neuronal performance during aging has still not been fully elucidated despite notable advances in our understanding of how the brain ages. While there are many behavioral assays that can be used to tease out gene effects during aging, the development of additional methods, such as those capable of measuring behavioral changes at a seconds scale, would provide potent tools to further enhance our understanding of mechanisms of neurotransmitter changes and their effect on aging. Here we used the MultiWorm Tracker and a series of analytic methods to determine the effect of subtle changes in locomotion that we would have missed by measuring average speed alone, the typical measure of changes in locomotion. And we suggest that these assays would likewise be useful for others who seek a more sensitive measure to changes in gene function.

Moreover, we describe a set of point mutations in Vacht which act as an allelic series with respect to severity of the mutations. By expressing the same amount of WT VAChT (through the introduction of a rescue WT Vacht mini gene P[Vacht]) to each allele and by balancing each allele over a deficiency that spans the Vacht locus, we were able to generate a partially rescued Vacht mutation series that circumvents lethality and use those lines to study the effect of altered Vacht function. We report that reductions in VAChT function leads to measurable defects in locomotion performance. Furthermore, we have developed locomotion-based methods to help capture a nuanced set of behaviors that would have been difficult to characterize otherwise. Importantly, we suggest a system to measure the effect of changes in cholinergic neurotransmission on behavior. Moreover, we report the finding that the VAChT function may be required for the proper navigation of animals in an open arena, which to our knowledge represents a novel role for that transporter.

Decrease in Vacht alters lifespan

Reports by others have shown that VAChT overexpressed in aging mice leads to increased neuromuscular aging and cholinergic dysfunction, and increased age decline in locomotion (Sugita et al., 2016). We have also shown elsewhere that VAChT overexpression leads to decreased lifespan (Showell et al., 2020). Based on these findings, we were surprised that a reduction in Vacht function also caused reduced lifespan. However, the nature of these mutations provided important context regarding how VAChT may affect lifespan. We found that two types of phenotypes, the severe mutant alleles Vacht1 and Vacht2 produced a stronger effect on lifespan; while the weak hypomorph Vacht8 had a phenotype similar to WT, suggesting that state of the function or expression of Vacht is related to its effect on lifespan and behavior. Importantly, with respect to cholinergic tone, subtle changes appear to have little to no effect on survivorship while strong changes in Vacht produce a strong effect. Therefore, there is an argument that survivorship is sensitive to changes in cholinergic tone with varying levels of Vacht producing corresponding effects on organismal lifespan. We note that we are unable to conclude definitively that allele severity correlates with decreased lifespan since we do not show an intermediate allele between the severe mutants and the hypomorphic allele. Overall however, the findings support the idea that changes in cholinergic neurotransmission can disrupt the normal aging process.

An allele-dependent effect of Vacht on baseline locomotion

The effect of decreases in VAChT function on locomotion has been well documented in different model systems (Kitamoto et al., 2000; Prado et al., 2006; Showell et al., 2020); and in each case, reduced Vacht expression impairs locomotion. Our findings expand upon these reports. Although our studies were performed in adults, the findings are in agreement with the effects reported previously (Kitamoto et al., 2000) in which homozygous Vacht2 mutant larvae decreased locomotion. Accordingly, in young flies, Vacht1-Res showed a significant reduction in locomotion behavior while Vacht2-Res showed a more modest impairment. Interestingly, Vacht8-Res did not have any detectable locomotion phenotype in young flies, suggesting that Vacht8-Res represents a minor disruption in the function of the protein. We note that in our measurement of average speed over a four-week period, our results in the WT largely mirrors that of others (Simon et al., 2006) in which there is a decline in locomotion across the lifespan in WT animals. In contrast to WT, however, the Vacht1-Res maintained remarkably stable, yet reduced locomotion rates, and it is conceivable that this is due to a limit to the contribution of cholinergic signaling to locomotion ability. By contrast, Vacht8-Res showed increased locomotion until week 4 in which flies appeared to move even faster than WT and overall showed a different speed trajectory compared to WT. We conclude that the Vacht8 mutant allele is a weak hypomorph.

Our work provides support for the robustness of average speed as a measure of locomotion and points to its limitations. For instance, since average speed, by definition, disregards the dynamics of locomotion during the assay, other relevant features such as trends and patterns, can be concealed and therefore go unnoticed. And indeed in our studies, we found this to be the case. By analyzing speed over the entire 60 seconds assay duration revealed that the reduced state of Vacht1-Res mutants was generally uniform over the course of the assay with little internal deviation which is in sharp contrast to Vacht8-Res which has a more varied profile. Similarly, we found that density and speed/time plots were particularly useful in providing a visualization of the data such that the speed dynamics becomes apparent in a way that average speed does not. Both assays confirm the findings on the average speed that Vacht1, the more severe mutation, maintained a strongly impaired speed/time profile throughout the assay period and the animal showed the highest speed density at very low speeds. By contrast, Vacht8-Res mutants have a higher speed profile that is gradual throughout the lifespan, but could be detected on a subpopulation of the flies at wk0 when changes on the average speed were not yet significantly different from the WT.

Overall, we detect an allelic series effect in the Vacht mutants such that there are clear distinctions among the three partially rescued Vacht mutants with respect to locomotion: Vacht1-Res shows severe deficit which is maintained through the period of the lifespan that we studied; Vacht2-Res is like WT and does not show an apparent defect; while Vacht8-Res moves faster than WT particularly in the latter weeks of the assay. Taken together, these three distinct phenotypes correlate with the severity of the mutation. Other investigators have also reported a link between the degree of deficits in Vacht expression and the resulting phenotype such that a 50% knockdown of Vacht produced a CNS phenotype while a more severe 70% knockdown of Vacht caused both CNS and neuromuscular junction (NMJ) effects (Prado et al., 2006).

Disruption in Vacht expression or function leads to centrophobism or thigmotaxis-like effects

The effect of cholinergic neurotransmission on the regulation of locomotion is well known (Kitamoto et al., 2000; Prado et al., 2006; Showell et al., 2020), however, its precise role in a behavior displayed by animals in an open field, where they tend to avoid the center (centrophobism) and move along the edge of the arena (thigmotaxis) is not clear. This behavior is observed in evolutionarily diverse organisms including zebrafish, and rodents and is thought to be a self-defense mechanism (Blazevic et al., 2012; Richendrfer et al., 2012). Previous report has depicted the role of serotonin in this behavior. In rats, increased 5HT concentration leads to an increased presence around the periphery (Blazevic et al., 2012). Moreover, studies in 5HT transporter KO mice showed strong thigmotaxis-like behavior (Kalueff et al., 2007). In contrast to serotonin, there is no clear role for cholinergic signaling in centrophobism or thigmotaxis. Here we show that unlike WT animals which show a slight preference for the periphery in an open arena, and whose behavior is unchanged regardless of the stage in the lifespan measured, each of the partially rescued Vacht mutants localizes to the periphery of the assay plate in a circular pattern (Figure 5). We used a heat map to show that the areas of greatest density are slightly weighted toward the periphery in the WT but oriented more strongly around the edges in the partially rescued Vacht mutants. What is most striking about these observations is that all of the partially rescued Vacht mutants, regardless of severity, show a similar pattern of migration, and the behavior is recapitulated throughout the different points in the lifespan measured. These findings are in sharp contrast to findings from the studies of locomotion rate in which the extent of the disruption in locomotion is dependent of mutation severity. We suggest that this report represents to our knowledge the first demonstration of a role in the CNS for ACh in regulating thigmotaxis-like behavior in Drosophila, and one of the first such reports of an involvement of ACh in controling locomotion patterns in an open field arena.

The literature on centrophobism or thigmotaxis as a measure of anxiety-like behavior in rodents is well established (Higaki et al., 2018; Simon et al., 1994). However, no such link has been similarly established in Drosophila. Despite the fact that both WT and Vacht mutants apt to move along the edge in a circular chamber, this tendency is exaggerated in the mutants. Importantly, defects in ACh signaling in the brain has been associated with anxiety-like behaviors in mice (Janickova et al., 2017; Piri et al., 2012), although the extent to which centrophobism or thigmotaxis is driven by anxiety is not clear (Martyn et al., 2012). It is conceivable that the behavior we observe in the Vacht mutants is related to self-defense mechanisms. As such, the effect of Vacht gene function on centrophobism or thigmotaxis could serve as a paradigm for investigating a possible role for ACh in regulating self-defense behavior in Drosophila.

It is also important to discuss this finding in the context of a requirement of ACh in different behavioral circuits. It is possible that the normal migration behavioral circuit requires definitive level of ACh and that any disruption that alters this level in turn affects the locomotion patterns in an open field. In support of this idea, we find that overexpression of VAChT also leads to a centrophobism or thigmotaxis-like phenotype that is similar to the one we observed in the partially rescued Vacht mutants. We note that we observed this effect in ~1 week old flies and therefore we cannot comment on how this effect will unfold over the course of weeks as we have done with the Vacht mutants. Interestingly, these Vacht overexpressing flies do not display any obvious deficits in average locomotion speed (Showell et al., 2020). And we conclude from our data that, at least in young flies, the ACh role in centrophobism or thigmotaxis is insensitive to changes in cholinergic tone and rather may be influenced by an ACh balance such that perturbation in Vacht expression or function is sufficient to disrupt the migratory behavior in Drosophila and that a neurological circuit that regulates locomotor patterns in an open field is distinct from the one that controls baseline locomotion rates. These findings could set the stage for future efforts aimed at understanding of how changes in ACh regulation on neurons regulate key behavioral functions and help tease out requirements for acetylcholine on distinct behavioral paradigms.

CONCLUSION

Overall, this study suggests that decreasing the amount of ACh released into the synaptic cleft by reducing Vacht function produces a graded effect on ACh-linked behaviors in adult Drosophila. And this effect is dependent on the behavioral circuit studied; circuits like the one that regulates locomotion and lifespan are sensitive to the severity of the mutation. Whereas the regulation of migration ability with an arena is not dependent on the severity of the disruption in VAChT function. This work benefited from the analytical methods that we developed to quantify at high resolution details of locomotion and identify a potentially novel function for ACh in the regulation of neurological function. And it serves to complement existing analytical tools that have been developed by others such as the Ctrax (Branson et al., 2009). We suggest that these methodologies could be deployed to further understand the contribution of ACh and perhaps other relevant neurotransmitters to the neuronal control of behavior.

HIGHLIGHTS.

  • Deficits in Vacht function cause an allele dependent effect in organismal survival

  • Demonstration of involvement of acetylcholine in thigmotaxis-like behavior

  • Differential role for acetylcholine signaling in regulating specific behaviors

  • First report of a weak hypomorphic Vacht point mutant allele in Drosophila

ACKNOWLEDGEMENTS

The authors wish to thank David Krantz (UCLA) for critical feedback on the conceptual design of the project.

Funding Sources: This work was supported by the following grant sources: NIH/NIGMS COBRE grant (4P20GM103653–05); and the NIA K01 grant (5K01AG049055–04)

Footnotes

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