Neuronal control of locomotor handedness in Drosophila

Abstract

Genetically identical individuals display variability in their physiology, morphology, and behaviors, even when reared in essentially identical environments, but there is little mechanistic understanding of the basis of such variation. Here, we investigated whether Drosophila melanogaster displays individual-to-individual variation in locomotor behaviors. We developed a new high-throughout platform capable of measuring the exploratory behavior of hundreds of individual flies simultaneously. With this approach, we find that, during exploratory walking, individual flies exhibit significant bias in their left vs. right locomotor choices, with some flies being strongly left biased or right biased. This idiosyncrasy was present in all genotypes examined, including wild-derived populations and inbred isogenic laboratory strains. The biases of individual flies persist for their lifetime and are nonheritable: i.e., mating two left-biased individuals does not yield left-biased progeny. This locomotor handedness is uncorrelated with other asymmetries, such as the handedness of gut twisting, leg-length asymmetry, and wing-folding preference. Using transgenics and mutants, we find that the magnitude of locomotor handedness is under the control of columnar neurons within the central complex, a brain region implicated in motor planning and execution. When these neurons are silenced, exploratory laterality increases, with more extreme leftiness and rightiness. This observation intriguingly implies that the brain may be able to dynamically regulate behavioral individuality.

Keywords: behavior; central complex; circuit mapping; individuality; personality.

Fig. 1.

Individual flies exhibit biases in left-right turning. (A) Schematic of a device for assaying left-right turning in individuals. Flies were placed into an array containing many individual Y-mazes. The mazes were illuminated from below and imaged from above, and the positions of the flies were recorded. (B) Detail of Y-mazes containing individual flies. (C) One hundred example turn paths through the Y-maze recorded from a single fly over 2 h (blue). Other colors highlight individual turns. (D) Left and right turn sequences for example flies of varying turn biases. Magenta ticks indicate left turns; green, right. (E) Observed distribution of turn bias scores (fraction right turns) measured from WT lines (solid lines), and corresponding expected distributions of turn bias scores (dashed lines). Sample sizes given in F. BK, Berlin-K; CA, Cambridge-A; CS, Canton-S. (F) The breadth of the distribution of turn bias scores for seven lines as measured by MAD. Error bars are ±SE estimated by bootstrap resampling. Dashed lines indicate MADs expected under a binomial null model. All lines other than w¹¹¹⁸ (transgenic background line) are nominally WT.

Fig. 2.

An individual's handedness is persistent over time. (A–C) Turn scores from individual flies measured in sequential experiments. Flies were assayed in the Y-mazes, stored individually, and then assayed a second time 1 d (A), 2 d (B), or 4 wk (C) later. (D) Correlation coefficient (r) of turn bias scores across flies tested in the Y-maze, stored individually, and then tested a second time either 1 d, 2 d, 1 wk, 2 wk, or 4 wk later. Error bars indicate ±SE as estimated by bootstrap resampling. n = 85 to n = 184 for all time points. (E) Mean turn bias of parental (brown) and F₁ generations (tan) derived from strongly biased CS individuals (gray bars). n indicates number of F₁s assayed. h² indicates estimated heritability. The dashed line indicates 50%. Error bars are ±1 MAD, as a measure of variability rather than error. F₁ and parental distributions are statistically indistinguishable.

Fig. 3.

The central complex regulates variability in turn bias. (A) The degree of variability in handedness for seven fly lines carrying mutations that disrupt the development of the central complex (purple bars). Three mutations significantly increase the MAD of the distribution of turn bias scores compared with heterozygous controls (gray bars). **P < 0.01, ***P < 0.001, as estimated by comparing bootstrap resampling of MAD values (15), Bonferroni corrected for multiple comparisons. Error bars are ±SE estimated by bootstrap resampling. Numbers indicate sample sizes. Purple boxes indicate neuropils grossly disrupted by each mutation. (B) Turn score variability (MAD) of lines with c465-GAL4 driving expression of temperature-sensitive modulators of neuronal activity (GAL80^ts;Kir2,1, Shibire^ts, dTRPA1, and control lines) at 23 °C (blue bars) and 33 °C (orange bars) temperatures. Error bars and P values as in A. (C–E) Max fluorescence z-projections of c465-driven expression of membrane localized (mCD8) or presynapse localized (nSyb) GFP (cyan), within the central brain (C) and central complex (D and E). Red counterstain is actin. Diagram indicates anterior-posterior extent of z-projection. Cal, mushroom body calyx; PB, protocerebral bridges; No, noduli; EB, ellipsoid body; d/vFB, dorsal/ventral fan-shaped body; PFN, PB-FB-No neurons.

Fig. 4.

PFNs regulate variability in turn bias. (A) Maximum fluorescence z-projections of R16D01-GAL4–driven expression of membrane localized mCD8::GFP (cyan), within the central complex. Red counterstain is actin. Diagram indicates anterior-posterior extent of z-projection. (B) As in A for the R73D06-GAL4 driver. (C) Lateral views of CD8::GFP driven by R16D01, R73D06, and c465-GAL4. PB, protocerebral bridges; FB, fan-shaped body; No, noduli; EB, ellipsoid body. d, e, f, layers of the ventral fan-shaped body, 1, 2, 3, 4, domains of the noduli. (D) Turn score variability of lines with various GAL4 lines driving shibire at 23 °C (blue bars) and 33 °C (orange bars). Bars are ±SE estimated by bootstrap resampling. Numbers indicate sample sizes. *P < 0.05, ***P < 0.001. Red boxes indicate PFN subtypes with high GAL4 expression; pink boxes indicate lower expression.