Cellular mechanisms for integral feedback in visually guided behavior

Results

We performed whole-cell patch-clamp recordings from HS cells during tethered flight (Fig. 1 A and B). At the start of flight, we recorded an increase in membrane potential from −42.2 mV (±0.07 mV SEM) to −40.1 mV (±0.12 mV SEM), an effect that was similar in magnitude to what has been observed in vertical system (VS) cells (20, 21). To directly compare behavioral and neuronal responses, we first presented a square-wave grating moving horizontally at different velocities. In between stimulus presentations, the flies performed closed-loop stripe fixation, which typically induces longer flight bouts. An example trace of an HS cell recording with the simultaneously measured behavioral output during flight is shown in Fig. 1C. For comparison, a response of the same HS cell during quiescence (i.e., when the animal was not flying) is shown below. Both the membrane potential of HS cells in the right hemisphere and the difference in wing stroke amplitude of the left and right wing (L-R) increase in response to rightward motion. However, flies occasionally perform fast turns in the opposite direction of stimulus motion, which probably correspond to body saccades in free flight (22, 23). Remarkably, these internally generated turns coincide with brief changes in the membrane potential of HS cells (indicated by the arrowheads in Fig. 1C). This effect might serve to counteract the response of HS cells to the visual stimulus that would normally be elicited by an active flight maneuver. Whereas this phenomenon was a consistent feature of HS cell physiology during flight, it was not the focus of this study and we have ignored these events in our subsequent analysis.

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Fig. 1.

Simultaneous neuronal and behavioral recordings. (A) Schematic of the setup for whole-cell patch-clamp recordings during flight. (B) Maximal intensity projection of the Gal4 line R27B03 crossed to UAS-eGFP showing the dendrites of three labeled HS cells of one lobula plate (approximately 30 µm in depth) in green. Neuropil staining shown in purple. (Scale bar: 50 µm.) (C) Example traces obtained from one fly. Periods of closed-loop stripe fixation are interspersed with 3 s of open-loop stimulus presentation (shaded gray areas), when a square wave pattern drifts horizontally in the cell’s preferred direction (PD) (here at 0.5 Hz temporal frequency). The traces indicate the optomotor behavior measured as difference between wing stroke amplitudes (L-R), the membrane potential (V) of an HS cell recorded simultaneously, and the response of the same HS cell to a similar stimulus during quiescence. HS cells depolarize in response to PD motion and hyperpolarize in response to null direction (ND) motion (Fig. S1). The motor output shows a similar directional dependence. Rightward stimulus motion elicits increases in L-R, corresponding to a right turn. During the closed-loop portion of the flight trials, the cells responded robustly to the horizontal motion of the stripe. Flies occasionally perform fast turns in the opposite direction of stimulus motion. These internally generated turns coincide with brief changes in the membrane potential of HS cells (purple arrowheads). (D) Mean and SEM (gray envelopes) of behavioral (L-R, green) and neuronal responses during flight (blue) and quiescence (black) of 11 flies for temporal frequencies of 2 and 7 Hz. Light gray areas indicate time of stimulus motion. (E) Mean and SEM of steady-state HS cell responses during quiescence (black) and flight (blue) and of behavioral responses (green), calculated during the second second of open-loop stimulus presentation. ND responses (Fig. S1) were subtracted from PD responses.

We found evidence for temporal integration in the optomotor system when comparing the time courses of HS cell activity and steering responses elicited by a square wave grating moving horizontally at different velocities. At low temporal frequencies (i.e., low stimulus velocities), both the behavioral turning responses (L-R) and the change in membrane potential of HS cells remain elevated throughout stimulus presentation (Fig. 1D, Left). At high temporal frequencies, however, HS cells exhibit a transient depolarization followed by adaptation to a low steady-state level, whereas the behavioral responses remain constant throughout stimulus motion (Fig. 1D, Right). During quiescence, HS cells exhibit even stronger adaptation (Fig. 1D, black traces). Based on these data, we constructed steady-state temporal frequency tuning curves by subtracting the steady-state responses (measured as the mean response during the interval of 1–2 s of stimulus presentation) to preferred and null direction motion (PD and ND, respectively; Fig. 1E). Whereas the HS cell tuning curve shows a distinct maximum, the behavioral tuning curve is quite broad. Differences in the shapes of these tuning curves have been attributed to the effects of neuromodulation on the motion vision pathway during flight (19). However, the temporal frequency tuning curve of HS cells measured during flight does not resemble the behavioral tuning curve. Instead, we propose that the broad plateau of the behavioral tuning curve is evidence for a leaky temporal integrator in the optomotor pathway. For long stimulus durations, the behavioral output asymptotes to approximately the same maximum value at all temporal frequencies and, thus, does not reflect the current value of the membrane potential of HS cells, but rather its short-term history.

To obtain direct estimates of the temporal filtering properties of both the behavior and the HS cell response, we determined their impulse responses by using an m sequence (24, 25). An m sequence is a pseudorandom binary sequence controlling, in this case, the instantaneous velocity of the pattern (Fig. 2A, Top). The pattern was a sine wave grating moving one pixel (2.25°) in PD for every 1 and one pixel (2.25°) in ND for every −1. For electrophysiological experiments, a 10th-order m sequence was presented at frequencies between 35 and 80 Hz. Convolving the stimulus with the HS cell responses during flight (Fig. 2A, Middle) and the simultaneously measured turning responses (L-R) (Fig. 2A, Bottom) gives an estimate for the impulse response of the cell and the behavior (Fig. 2B and Fig. S2). The impulse response of HS cells rises sharply and decays to zero in less than 400 ms (Fig. 2B, blue line). The decay of the neuronal response is well approximated by an exponential with a time constant of 65 ms (least square fit, R = 0.936). Because the behavioral response during physiological recording exhibited a longer time constant (Fig. S2), we obtained additional behavioral data from intact wild-type flies (without recording neuronal responses) by using a longer (15th order) m sequence. The behavioral impulse response is broader than that of the HS cells and decays with a much slower time course (Fig. 2B, green line). The decay of the behavioral response is well approximated by the sum of two exponentials with time constants of 0.52 s and 4.44 s (least square fit, R = 0.996). The transfer function with which the neuronal response would have to be convolved to produce the behavioral response (Fig. 2B, red line) accordingly has an initial peak and a very long tail and is thus suggestive of a leaky integrator between HS cells and motor output.

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Fig. 2.

HS cell and behavioral impulse responses. (A) Example trace of an m-sequence experiment. An m-sequence controls the instantaneous horizontal velocity of a large field sine grating. +1 (−1) indicates that the stimulus was moving in the PD (ND) of the cell. The change in membrane potential (V) of one HS cell and the turning response (L-R) elicited by this stimulus are shown below. (B) Mean neuronal (blue, n = 5) and behavioral (green, n = 4) impulse responses obtained from m-sequence data. SEM is indicated in gray. Behavioral data were obtained from intact flies by using a longer m-sequence than for neuronal data. The red line is the transfer function (TF) between the two responses.

Because behavioral responses to the m-sequence stimulus are relatively small, we performed a further test for the presence of integration in the horizontal motion pathway. We presented flies with a grating moving horizontally at a temporal frequency of 7 Hz across a range of stimulus durations and measured behavioral responses of intact flies during the stimulus motion and the subsequent 3 s during which the stimulus was stationary (Fig. 3A and Fig. S3). Evidence for an integral term in the sensory-motor system was manifest by two features of the behavior: a rise in the peak motor response with longer stimulus durations (Fig. 3E, green trace) and a very slow decay of the turning response after the stimulus motion ceased. The persistence of the motor response following the offset of visual motion is particularly striking, given the brevity of steering maneuvers in freely flying flies (26).

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Fig. 3.

Behavioral and HS cell responses to motion stimuli of variable duration. (A) Mean behavioral responses of 15 intact wild-type flies to a large-field pattern rotating in the PD for a variable length of time (from 0 to 2 s, indicated by the horizontal bars above) and then remaining stationary for 3 s. Before the stimulus, flies performed closed-loop stripe fixation. (B) Mean membrane potential (V) of HS cells during flight in eight flies in response to the same stimuli. (C) Prediction of the fluorescence signal based on reported time constants of GCaMP6f estimated by convolving baseline-subtracted neuronal responses in B with the kernel shown in G. Predictions were normalized to the peak response of the shortest duration trial. (D) Measured GCaMP6f fluorescence changes (ΔF/F) in the terminals of HS cells of 21 flies during flight in response to similar stimuli as in A and B. (E) Mean and SEM of behavioral responses (green solid line) and peak predicted (dotted lines) and measured (solid lines) fluorescence changes during flight (blue) and quiescence (black) from A, C, D, H, and I plotted against stimulus duration. The baseline (mean during 0.5 s before open-loop stimulus) was subtracted from the measured GCaMP6f responses. (F, H, and I) Same as B–D, but during quiescence. (G) Kernel for predicting fluorescence changes purely based on GCaMP6f dynamics. Time constants for rise and decay (τ_0.5) are 50 and 490 ms, respectively, and were chosen to match the prediction to the time course of the imaging data for the shortest duration stimulus (0.1 s) in D.

The membrane potential responses of the HS cells do not, however, resemble a temporal integration of the stimulus input (Fig. 3 B and F). During quiescence the responses are nearly identical for all stimulus durations, consisting of a brief transient at the onset of motion followed by a rapid decay (Fig. 3F). The membrane potential response more closely resembles a differentiation of the visual motion input than integration. During flight, the cells respond with a transient depolarization of the same peak magnitude, but followed by a small plateau response that lasts for the duration of the stimulus (Fig. 3B). The discrepancy with the behavior is somewhat puzzling given that HS cells are thought to be only a few synapses upstream of the motor neurons driving wing motion.

One possible explanation for this discrepancy is that the membrane potential of an HS cell does not provide an accurate measure of its synaptic output. Assuming that some HS cell connections are mediated by chemical synapses, the calcium levels in the terminals of the neurons could have different dynamics and provide a mechanism for temporal integration. To test this hypothesis, we expressed the genetically encoded calcium indicator GCaMP6f (27) in HS cells (Fig. S4) and performed a set of two-photon imaging experiments during flight with a protocol similar to that used during whole-cell recordings. In contrast to the membrane potential responses, the GCaMP6f signals from HS cell terminals are consistent with the behavioral results in that they increase with increasing stimulus duration and decay very slowly after the end of motion (Fig. 3D).

The fluorescence changes we measured in the HS cells are determined by the time history of free calcium within the cell and the dynamics of GCaMP6f binding. To estimate the influence of indicator binding dynamics on the signals, we constructed a GCaMP6f response kernel based on our data (Fig. 3G). The rise and decay time constants were chosen such that a convolution of the kernel with the membrane potential response to the shortest duration stimulus best matched the measured GCaMP6f response during flight. The time constants obtained in this manner were quite similar to reported on and off rates of GCaMP6f (27). Based on this consistency, we predicted the fluorescence responses to different stimulus durations by convolving the GCaMP6f kernel with the electrophysiological responses to those stimuli. The resulting predictions should represent a conservative estimate for how the temporal dynamics of the indicator filter the actual calcium changes in HS cells. In the case of a quiescent fly, our model predicts that the fluorescence changes induced by visual motion should change little with increasing stimulus length (Fig. 3H). The actual measured GCaMP6f responses, however, grow measurably with stimulus duration (Fig. 3I). This discrepancy suggests that nonlinear processes within the HS cells strongly magnify small differences in membrane potential and allow calcium to accumulate in the terminals according to a time scale that cannot be explained by the binding rates of the indicator.

The predicted and measured GCaMP6f signals from flying flies differ from the quiescent case in several ways. First, the magnitude of the measured GCaMP6f signals in HS cells is substantially larger during flight, to a degree that is not proportional to the differences in membrane potential. Second, based on convolving our estimated indicator kernel with the measured membrane potential responses, we predict that the GCaMP6f signal should rise with increasing stimulus length. This growth of the predicted response curves with stimulus duration is due to the low-pass filtering characteristics of the indicator acting on the small but consistent plateaus of the membrane potential during flight (Fig. 3B) that are not present during quiescence (Fig. 3F). However, the predicted responses during flight severely underestimate the magnitude and long decay of the measured GCaMP6f signals, as was the case during quiescence (except for the shortest duration trial used to fit the kernel parameters). The failure of the model provides evidence of calcium accumulation within the terminals of the HS cells. Furthermore, the measured GCaMP6f signals during flight (Fig. 3D) bear a strong resemblance to the behavioral output (Fig. 3A).

To better illustrate the degree of integration, we plotted peak responses as a function of stimulus duration for the predicted and measured GCaMP6f data (Fig. 3E, blue and black traces). GCaMP6f signals in the terminals of the HS cells rise with stimulus duration until saturation in a manner that represents an integration of the visual motion input and closely matches the optomotor behavior. The integration timescale imparted by the indicator dynamics falls short of predicting the strong increase of signal with increasing stimulus duration. It is also informative to compare the results of quiescent and flying flies. Not only are the GCaMP6f signals smaller during quiescence, they do not exhibit the degree of integration during flight as indicated by the shallower slope and earlier saturation.

The prolonged response plateaus in HS cells recorded during flight, although small as measured in the cell body, might be important for generating the calcium accumulation in the terminals. These plateaus are absent during quiescence, which correlates with the reduced accumulation of calcium. The appearance of these plateaus during flight might result from modulation of intrinsic HS cell properties or alternatively could reflect changes in their synaptic drive. To address this question, we expressed GCaMP6f in T4 and T5 cells that provide directional motion information to LPTCs (9, 10) (Fig. 4A). We measured fluorescence changes in the HS cell layer of the lobula plate in response to a similar set of horizontal motion stimuli of different durations during quiescence and flight. In contrast to the previous experiments, we held the square-wave grating stationary during the segments between motion trials, because in initial experiments, T4 and T5 cells exhibited a strong response to just the presentation of the stationary grating, which made it difficult to discriminate responses to motion of different durations if the pattern started moving immediately after appearing. During quiescence, responses to all different durations of motion are very similar except for a small depolarization after the end of motion for the longer duration trials (Fig. 4B, lower traces). During flight, however, we observed a quite large increase in baseline fluorescence and an increase of the response to visual motion with increasing stimulus duration (Fig. 4B, upper traces). For all durations, the GCaMP6f signal exhibited a fast initial rise followed by a slow increase throughout stimulus motion. This increase in GCaMP6f response for longer stimulus durations during flight in the T4 and T5 cells could explain the prolonged plateau potentials in postsynaptic HS cells, which are present during flight but absent during quiescence. These results indicate that at least some of the changes in HS cell physiology caused by flight are due to modulation of upstream circuits. The slow rise in the GCaMP6f signals in the T4 and T5 cells during flight might also be interpreted as evidence for weak integration. However, the rise in fluorescence with increasing stimulus duration in the T4 and T5 cells is much slower and smaller than that observed in the HS cell terminals using the same calcium indicator, especially for the short duration trials.

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Fig. 4.

T4 and T5 cell responses to stimuli of varying durations similar to Fig. 3. (A) Mean image of T4 and T5 cell terminals in the lobula plate obtained during recording from one fly with the region of interest circled in red and the background in green. In this image, medial would be on the top, lateral on the bottom, dorsal to the right, and ventral to the left. The region of interest was chosen to match the layer of HS cell dendrites, which is the deepest layer (in this view most medial) next to the lobula, where T5 cells have their dendrites. (B) Fluorescence changes in the HS cell layer during quiescence (lower traces) and flight (upper traces) of 10 flies. The grating was held stationary in between trials and started moving at time 0 for the time indicated by the colored bars on top of the graph.