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compartments.

These insights have led to the first optical recordings of directional selective responses from neurons in fruit flies using the new troponin-based GECI TN-XXL.

4.3 Directional Selective Motion Responses in LPTCs

LPTCs have been extensively studied in blowflies (see 1.1). Imaging experiments revealed calcium currents in dendritic and axonal compartments of VS, HS, and CH cells e.g. [158, 12, 159, 160].

In two preliminary experiments I present here the first calcium measurements that demonstrate direction selectivity in VS cells of fruit flies using a novel genetically encoded probe. Axons of four different, yet unidentified VS cells showed steady-state responses of around 20 % ∆R/R. A dendritic compartment showed a transient, directional selective motion-on-response. These responses are reminiscent of transient calcium responses ex- hibited in higher order dendritic branches of a VS1 cell in response to a locally restricted moving grating [161]. It has been shown, that local motion responses in the dendrites of LPTCs are integrated in the axons [12]. However, the stimulus used here was not lo- cally restricted but a large field grating. Thus, the local, motion induced calcium response should last throughout preferred direction motion stimulation. From former studies on blowflies local calcium signals in higher order branches of LPTC dendrites are expected to fluctuate with the temporal frequency of the stimulus grating [12]. In other studies on blowflies it was shown that synaptic input from local elementary motion detectors to VS cell dendrites may come through GABA receptors and nicotinic acetylcholin receptors [162, 163, 164]. The latter shape the local calcium fluctuations. Integrated axonal calcium signals are shaped by voltage gated calcium channels [159, 164].

In the presented experiments, the contrast frequency of the stimulus grating was roughly 1 Hz and motion was presented for 5 s in preferred and null direction. Thus, a local response to a single bar within the grating can also not account for the transient response. Behavioral experiments, measuring optomotor responses suggest a maximum response at 1

Hz in fruit flies [19]. The transient on-response found in the VS cell dendrite can thus not be explained by stimulus properties. Furthermore, the response decline is fast. Adaptation- like processes that might account for this response decay have not been described [165]. It will be interesting to see whether this transient response will be confirmed in further experiments with more precisely defined stimulus conditions.

The fluorescence signal measured with TN-XXL at VS cell axons during preferred direc- tion motion reached ≈ 20% ∆ R/R in amplitude at steady-state. Extrapolating from the experiments at neuromuscular junctions, this relates to an increase of>100nM in axons. CCD-camera-based calcium imaging in blowflies yielded fluorescence change amplitudes of up to 10 % ∆ F/F using calcium green, although with superior signal-to-noise ratio at 4

Hz sampling frequency [12]. Using 2-photon microscopy amplitudes of 50-100 % ∆ F/F were measured [14]. Calcium was not quantified in these measurements. Null direction motion did not induce decrease in fluorescence response in our measurements, as found in Calliphora [12]. This is likely due to a lack of the GECIs sensitivity below the resting calcium concentrations of the VS cells.

Recent characterizations of VS cells in Drosophila support the assumption that general features of VS cells are shared by fruit flies and blowflies (Maximilian Joesch, unpublished observations): The 6 VS cells found in Drosophila are directional selective. They hyper- polarize in response to downward motion and depolarize in response to upward motion. The centers of their receptive fields are distributed along the flies azimuth. The width of the receptive fields is broader than expected from the receptive field of their dendrite, indicative of possible electrical coupling of VS cells as shown in blowflies [14]. VS cells, unlike a speedometer, display a temporal frequency tuning to moving gratings. This means that if a sine grating of spatial wavelength A, elicits a maximum VS cell response at speed B, then a grating of wavelength 2xA will elicit a maximum response at speed 2xB, because both result in the same contrast frequency. In Drosophila the VS cells are best tuned to a contrast frequency of 1Hz. The velocity tuning is a major prediction, derived from the algorithmic model of a correlation type motion detector in action [5]. Also, the charac- teristic transient step-response shown in blowflies in agreement with the correlation-type motion detector model [166, 167] were reproduced in fruit fly VS cell recordings. Another prediction from the model, contrast invariance of the VS cell response to motion has also been demonstrated in blowflies and fruit flies (all: Maximilian Joesch, unpublished ob- servations). Moreover, as had been proposed for blowflies, VS cells of Drosophila receive synaptic input at their dendrite via GABA receptors and nicotinic acetylcholine receptors. GABA receptors were also found on the axon terminals (Shamprassad V. Raghu, personal communication and [13]).

Together with morphological evidence [7], the first electrophysiological recordings from fruit fly VS cells and histological data on synaptic distribution on VS cells indicate that the motion detection system of flies is evolutionarily conserved between species. Thus, the rich