Dendritic Calcium Accumulation Associated With Direction-Selective Adaptation in Visual Motion-Sensitive Neurons In Vivo

Rafael Kurtz, Volker Dürr, and Martin Egelhaaf

Lehrstuhl für Neurobiologie, Fakultät für Biologie, Universität Bielefeld, D-33501 Bielefeld, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kurtz, Rafael, Volker Dürr, and Martin Egelhaaf. Dendritic Calcium Accumulation Associated With Direction-Selective Adaptation in Visual Motion-Sensitive Neurons In Vivo. J. Neurophysiol. 84: 1914-1923, 2000. Motion adaptation in directionally selective tangential cells (TC) of the fly visual system has previously been explained as a presynaptic mechanism. Based on the observation that adaptation is in part direction selective, which is not accounted for by the former models of motion adaptation, we investigated whether physiological changes located in the TC dendrite can contribute to motion adaptation. Visual motion in the neuron's preferred direction (PD) induced stronger adaptation than motion in the opposite direction and was followed by an afterhyperpolarization (AHP). The AHP subsides in the same time as adaptation recovers. By combining in vivo calcium fluorescence imaging with intracellular recording, we show that dendritic calcium accumulation following motion in the PD is correlated with the AHP. These results are consistent with a calcium-dependent physiological change in TCs underlying adaptation during continuous stimulation with PD motion, expressing itself as an AHP after the stimulus stops. However, direction selectivity of adaptation is probably not solely related to a calcium-dependent mechanism because direction-selective effects can also be observed for fast moving stimuli, which do not induce sizeable calcium accumulation. In addition, a comparison of two classes of TCs revealed differences in the relationship of calcium accumulation and AHP when the stimulus velocity was varied. Thus the potential role of calcium in motion adaptation depends on stimulation parameters and cell class.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Many sensory receptors and neurons adapt to a constant input or to repeated stimulation. Adaptation leads to a gradual decrease of response amplitude with time. This can reflect a shift in the operating range of the cell. Sensory systems thus retain their ability to detect small deviations of stimulus intensity from the adapting background level over a large range of stimulus intensities.

Decreased responsiveness on prolonged exposure to moving stimuli has been demonstrated in visual motion-sensitive neurons of insects and mammals (e.g., rabbit: Barlow and Hill 1963; cat: Vautin and Berkley 1977; fly: Maddess and Laughlin 1985; monkey: Petersen et al. 1985; butterfly: Maddess et al. 1991; wallaby: Ibbotson et al. 1998). Increased sensitivity to changes in velocity around an adapting level has been observed psychophysically in humans (Clifford and Langley 1996b; Clifford and Wenderoth 1999) and electrophysiologically in the fly's H1 cell (Jian and Horridge 1991; Maddess and Laughlin 1985). This neuron is 1 of more than 30 motion-sensitive tangential cells (TCs), which have been individually identified in the lobula plate, a part of the third visual neuropil (review articles: Egelhaaf and Borst 1993a; Hausen 1984; Hausen and Egelhaaf 1989). Movement detection is thought to be based on correlation of input from one retinal location with temporally delayed input from a neighboring location. A fully direction-selective element, being excited by motion in the preferred direction (PD) and inhibited by motion in the opposite direction ("null direction," ND), is obtained by subtracting the output signals of such a correlator from that of its mirror-symmetrical copy. This is called an "elementary motion detector," EMD (review articles: Borst and Egelhaaf 1989; Egelhaaf and Borst 1993b; Srinivasan et al. 1999). The vast dendrites of TCs are assumed to receive retinotopically arranged input from numerous local EMDs, thus being able to respond to certain types of optic flow in a large part of the visual field (Hausen 1981; Krapp and Hengstenberg 1996).

In the H1 neuron, motion adaptation remains restricted to that part of the receptive field in which an adapting stimulus was presented (Maddess and Laughlin 1985). Since no cell intrinsic local mechanism was known then, the associated physiological changes were thought to be located presynaptic to H1. Shortening of the EMD filter time constant, and thus tuning the motion detector to higher velocities, was proposed to underlie motion adaptation (Clifford and Langley 1996a; de Ruyter van Steveninck et al. 1986). A recent investigation on another class of TC, the horizontal system (HS) cells (Harris et al. 1999a), casts doubt on this model of motion adaptation: prolonged movement did not cause changes in the optimal velocity tuning as would be expected from adaptation of the EMD filter time constant. Therefore the physiological mechanism leading to motion adaptation and its location is still unclear.

In this study, we direct our attention to intrinsic physiological changes in the motion-sensitive TCs induced by prolonged stimulation. More specifically, we focus on a possible functional role of intracellular Ca2+. Because in many receptor cells and neurons the intracellular Ca2+ concentration affects cellular excitability, e.g., by regulating Ca2+ dependent conductances (review articles: neurons, Sah 1996; photoreceptors, Koutalos and Yau 1996; olfactory receptors, Menini 1999; hair cells, Fettiplace and Fuchs 1999), we investigate the relationship between adaptation and Ca2+ concentration changes and motion adaptation in TCs. Motion in restricted parts of the receptive field has been shown to lead to local Ca2+ accumulation, which remains restricted to the part of the dendrite getting input from the stimulated region of the receptive field (Borst and Egelhaaf 1992; Dürr and Egelhaaf 1999; Egelhaaf and Borst 1995). Hence, a Ca2+-based mechanism of motion adaptation in the TC could, similar to the formerly proposed presynaptic mechanism, account for the localized adaptive characteristics.

A Ca2+-based mechanism would further imply that motion adaptation is direction selective. This expectation results from the observation, that major increases in dendritic Ca2+ concentration of TCs are only elicited by motion in the PD but not in the ND (Dürr 1998; Egelhaaf and Borst 1995). However, motion adaptation has not been analyzed before with test stimuli moving in the ND because former studies were mainly based on extracellular recordings of H1, which ceases spiking during motion in the ND. In our study, motion adaptation is tested both in the PD and in the ND by recording intracellularly from HS cells, in which graded membrane-potential changes can be recorded even in the axon (Hausen 1976, 1982a,b). We investigate the relation of calcium concentration changes to the electrical response characteristics underlying adaptation in this cell class and, additionally, in another class of TC, the centrifugal horizontal (CH) cells (Eckert and Dvorak 1983; Egelhaaf et al. 1993; Hausen 1976). The latter cell class is supposed to possess an input organization similar to HS cells but to differ with respect to its output organization: its dendrites do not only form postsynaptic sites to EMDs but also dendritic presynaptic sites (Gauck et al. 1997) mediating inhibition to other TCs within the lobula plate (Warzecha et al. 1993). Therefore in the CH cell, Ca2+ might not only be relevant for modulatory functions but also for the control of transmitter release.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Electrical and optical recordings were performed in vivo from the three HS cells (Hausen 1982a,b) and the two CH cells (Eckert and Dvorak 1983; Egelhaaf et al. 1993; Hausen 1976) in female blowflies (Calliphora erythrocephala), aged 1-3 days. Data were pooled for each cell class because the major difference within a class is the receptive field location with known physiological differences being only of minor significance (Eckert and Dvorak 1983; Hausen 1982a,b). For information about the animal preparation and closer details concerning the further experimental procedures, see Dürr and Egelhaaf (1999). Data are presented as means ± SE.

Electrophysiology

Ringer solution (containing, in mM, 128.3 NaCl, 5.4 KCl, 1.9 CaCl2, 4.8 NaHCO3, 3.3 Na2HPO4, 3.4 KH2PO4, 13.9 glucose, pH 7.0; all chemicals from Merck) was used to prevent desiccation of the recording region and for filling a wide-tip glass pipette used as indifferent electrode. Electrodes for intracellular recording were made from borosilicate glass (GC100F-10, Clark Electromedical) on a Brown-Flaming puller (P97, Sutter Instruments) and had resistances of 20-40 MOmega when filled with 1 M KCl. Voltage records (Axoclamp 2A, Axon Instruments) were low-pass filtered at 2 kHz, and AID converted at a sampling rate of 1 kHz with an amplitude resolution of 0.0244 mV (DT2801A, Data Translation). Part of the recordings were performed during iontophoretic injection of the fluorescent dye by a weak tonic hyperpolarizing current (see following text). No significant differences in the electrical properties were observed between the cells hyperpolarized in this way and the cells recorded without current injection.

Imaging changes in intracellular Ca2+ concentration

The calcium-sensitive dye Fura-2 was injected iontophoretically into single neurons by a hyperpolarizing current of 0.8 nA, with electrodes containing (in mM) 33.3 KCl (Sigma), 1.7 KOH (Merck), 20.0 fura-2 pentapotassium salt (Molecular Probes), and 33.3 HEPES (Sigma), pH 7.3. Relative changes of intracellular ionic free Ca2+ concentration (Delta [Ca2+]i) were determined during or immediately following electrophysiological recording by a single wavelength measurement according to Vranesic and Knöpfel (1991). Epifluorescence measurements were performed under an upright microscope (Universal III-RS, Zeiss). Fluorescence was elicited at 380 nm (light emitted from a Hg arc lamp, HBO 100W, Osram, and band-pass filtered with a bandwidth of 10 nm) and measured after passage of a 410-nm dichroic mirror and a 500- to 530-nm band-pass filter. Images were magnified 20-fold by the microscope objective (UD20, Zeiss) and acquired with a CCD camera (PXL, Photometrics) controlled by PMIS software (GKR Computer Consulting). Acquisition rate was approximately 3 Hz and spatial resolution was 9 µm. Delta F/F was determined relative to an image obtained prior to visual stimulation. The values were averaged in a region covering the entire dendritic tree. Increases in [Ca2+]i lead to a decrement in fluorescence. For convenience when comparing the calcium signals with the electrical signals of a cell, the former were inverted in the figures. Increments in [Ca2+]i are thus indicated by positive Delta F/F values. Background fluorescence as determined for each image in a region surrounding the dendrite was subtracted from the fluorescence images.

Visual stimuli

TCs were stimulated by two stripe patterns projected through a circular aperture on a translucent hemisphere and moving horizontally in the cells' receptive field. The stimulus was presented ipsilaterally and was characterized by the following parameters: diameter, 66°; spatial wavelength, ca. 33°; contrast, 0.68; mean light intensity, 12 cd m-2 with a spectral composition of a Hg arc lamp (HBO 100W, Osram) filtered by a blue filter (BG1, Schott). The stimulus velocity depended on the protocol and is given in the text and figure legends as temporal frequency (cycles per second; 2-Hz temporal frequency thus corresponds to an angular velocity of 66° s-1).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Adaptation of TC responses

The HS and CH cells of the fly respond to visual motion with graded membrane potential changes (Delta EM). In HS cells, depolarizing responses are usually superimposed by spike-like rapid depolarizations (Hausen 1982a). When a stripe pattern in the receptive field starts to move at a constant velocity in the PD of the cell, the membrane potential of both HS and CH cells rapidly shifts to a depolarized state followed by a slow decline to a steady-state level (Fig. 1). This transient behavior can, in part, be attributed to the mechanism of motion detection itself (Egelhaaf and Borst 1989) but additionally reflects changes in the movement processing system dependent on stimulus history, i.e., motion adaptation (Maddess and Laughlin 1985). The influence of stimulus history becomes obvious if a test stimulus is preceded by an adapting stimulus: if the interval between these two stimuli is sufficiently short (e.g., 0.1 s, as in Fig. 1), the early component of the response to the test stimulus is attenuated, rendering the test response less transient than the adapting response. The latter can serve as reference in our experimental procedure (Fig. 1). Motion adaptation, measured as the attenuation of the early component of the test response, can also be observed in both HS and CH cells for stimuli moving in the ND.



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Fig. 1. Adaptation in the graded membrane potential responses of tangential cells (TCs). Membrane potential change (Delta EM) of a horizontal system cell (HS) (left) and a centrifugal horizontal (CH) cell (right) relative to the resting level (- - -, determined in a 1-s period prior to stimulation) in response to visual motion stimuli (marked by underneath the response traces). An adapting stimulus was followed by an identical test stimulus, separated by an interval of 100 ms. Stimulus motion lasted 2 s, with 2-Hz temporal frequency. Direction of motion was either in the preferred direction (PD, top) or in the null direction (ND, bottom) of the neuron. Individual traces, recorded from 1 HS and 1 CH cell, respectively, were averaged and smoothed off-line for presentation using a running average of 25 ms width. Time windows for calculation of the early response component (100-300 ms after stimulus onset) and the late response component (1-2 s after stimulus onset) are indicated by gray shading (). Delta R, denoting the difference between adapting and test response (indicated here for the early response components), was used as a measure of adaptation.

Direction selectivity of adaptation

Adaptation is weaker and recovery from adaptation is faster for motion in the ND compared with the PD. This was shown quantitatively for HS cells by presenting adapting and test stimuli, separated by variable intervals. The experiments were done both for motion in the PD and in the ND (Fig. 2, left). To allow easier comparison of Delta EM in response to PD motion (top traces in each row), and Delta EM in response to ND motion (bottom traces in each row), ND traces are shown inverted and scaled by a factor of 1.5. Direction dependency is most prominent at the medium interval length (0.5 s), where the attenuation of the early component of the test response is pronounced for PD motion but hardly detectable for ND motion.



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Fig. 2. Direction selectivity of adaptation and dendritic Ca2+ accumulation. Delta EM and concentration change of intracellular ionic free calcium (Delta [Ca2+]i) in response to adapting and test stimuli. Shown are averaged responses measured in vivo in HS cells with voltage and Ca2+ imaging performed simultaneously or with Ca2+ imaging performed independently using identical stimulation (the latter within about 0.5 h of voltage recordings). Stimulation protocol as in Fig. 1, except that 3 different inter-stimulus intervals were tested (top, 0.1 s; middle, 0.5 s; bottom, 4 s; stimulation periods are marked by horizontal bars underneath the response traces). For easier comparison, Delta EM in response to ND motion (left, bottom traces in each row) is shown inverted and scaled by a factor of 1.5. Averaged time course of Delta [Ca2+]i (right) determined as relative fluorescence change (Delta F/F) at lambda  = 380 nm in areas covering the whole dendrite of HS cells filled with Fura 2. The horizontal line represents the baseline given by the first image, in relation to which Delta F/F in all subsequent images was calculated. As explained in METHODS, Delta F/F values are inverted, with positive Delta F/F corresponding to increments in [Ca2+]i. Unlike the Delta EM traces, Delta F/F traces of PD and ND stimulation are drawn with the same orientation and scale. Sample sizes: electrophysiological data: n = 12 (same set of cells for PD and ND). Data on Ca2+: n = 5-6 for PD and n = 3-4 for ND.

In Fig. 3A, the amplitudes of the early component (200-ms period, starting 100 ms after stimulus onset, see Fig. 1) of adapting and test responses are plotted for the PD and the ND. Adaptation is marked by a weaker early component of the response to the test than to the adapting stimulus. For motion in the PD, this amplitude reduction is significant for the 0.1- and 0.5-s inter-stimulus intervals. No significant amplitude difference between adapting and test response can be observed if both are separated by a 4-s interval. In each cell tested, Delta R, i.e., the difference between the early component of the response to the adapting and to the test stimulus, respectively (see Fig. 1), is positive for both the 0.1- and the 0.5-s inter-stimulus interval (averaged values: Delta RPD = 4.08 ± 0.50 mV for the 0.1-s interval and Delta RPD = 2.63 ± 0.32 mV for the 0.5-s interval, n = 12). Statistical significance of the difference between adapting response and test response was confirmed by a Wilcoxon rank test (P < 0.005 for both 0.1- and 0.5-s inter-stimulus interval, n = 12). In contrast, an adapting stimulus in the ND leads to an attenuation of the early component of the test response (leading to negative Delta R values) in only 8 of 12 cells for both the 0.1- and the 0.5-s interval. Averaged for all cells, the absolute values of Delta R for the ND (Delta RND = -0.76 ± 0.40 mV for the 0.1-s interval and Delta RND = -0.45 ± 0.32 mV for the 0.5-s interval, n = 12) are almost an order of magnitude lower than the corresponding values for the PD (see preceding results). Hence for ND motion, a statistical difference between adapting response and test response is given only for the 0.1-s interval at a low significance level (Wilcoxon rank test, P < 0.1, n = 12).



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Fig. 3. Time course of recovery from adaptation and after-reactions following PD and ND motion. A: amplitude of the early component (top) and the late component (bottom) of Delta EM (for time windows, see Fig. 1) in response to adapting (open circle ) and test stimuli (), plotted as a function of inter-stimulus interval. Responses to adapting stimuli serve as reference. The strength of adaptation is thus indicated by the difference between adapting and test responses. Mean ± SE of 12 HS cells (same set of cells for PD and ND). B: after-reactions recorded from HS cells (averaged responses from 10 cells) following a 2-Hz motion stimulus of 2-s duration in PD (top) and ND (bottom). Time 0 marks the cessation of stimulus motion. Baseline voltage was determined in a 1-s period prior to stimulation. For presentation, traces were smoothed off-line using a running average of 10-ms width.

To assess the directional selectivity of adaptation, we determined the attenuation of the early response component by a relative measure (r = test response/adapting response). Ratios calculated for PD (rPD) and ND motion (rND), respectively, were statistically different both for the 0.1- and the 0.5-s inter-stimulus interval. Thus adaptation contains a directionally selective component (0.1-s interval: rPD = 0.645 ± 0.036, rND = 0.887 ± 0.074; 0.5-s interval: rPD = 0.762 ± 0.028, rND = 0.970 ± 0.085, H0: rPD = rND, Wilcoxon rank test, P < 0.05 in both cases, n = 12).

Regardless of the direction of motion (PD or ND), the late component of the test response (last second of the 2-s stimulation period, see Fig. 1) is not greatly diminished by adaptation (Fig. 3A). Nevertheless, probably due to the better signal-to-noise ratio resulting from the larger time window, for an inter-stimulus interval of either 0.1 or 0.5 s, there is significant attenuation of the late response for PD and for ND motion (0.1-s interval: Delta RPD = 0.57 ± 0.23 mV, Delta RND = -0.56 ± 0.14 mV; 0.5-s interval: Delta RPD = 0.65 ± 0.26 mV, Delta RND = -0.25 ± 0.10 mV, Wilcoxon rank test, P < 0.05 in all cases, n = 12). No direction selectivity was found for adaptation of the late response components (0.1-s interval: rPD = 0.945 ± 0.034, rND = 0.900 ± 0.034; 0.5-s interval: rPD = 0.923 ± 0.034, rND = 0.953 ± 0.020, 4-s interval: rPD = 1.106 ± 0.107, rND = 1.091 ± 0.044; H0: rPD = rND, Wilcoxon rank test, P > 0.05 in all cases, n = 12).

Since PD motion elicits, on average, stronger responses than ND motion, direction-selective effects could be mimicked by processes that are in fact nonlinearly dependent on response size. Therefore we also compared adaptation in HS cells with approximately equally sized PD and ND responses. As has been found for the whole cell sample, the attenuation of the early response component by adapting stimuli was more pronounced and longer lasting for PD motion than for ND motion in this subset of neurons. Thus adaptation in TCs has a genuine direction-selective component.

In conclusion, our results indicate that motion adaptation in HS cells is characterized by both direction-selective and nondirectional mechanisms. Direction selectivity is reflected in a more pronounced attenuation of the early component of the test response to PD motion compared with ND motion. Additionally, recovery from adaptation is faster for PD than for ND motion. Compared to direction-selective adaptation, the nondirectional component of motion adaptation is weak but does result in a statistically significant reduction of the late component of the test response.

Adaptation in CH cells, tested experimentally in the same way for a smaller cell sample, followed a similar pattern. This, however, does not preclude quantitative differences in the characteristics of adaptation between the two cell classes.

After-reactions reflect motion adaptation in its strength and recovery time course

After PD motion stops, the membrane potentials of HS and CH cells hyperpolarize, a phenomenon referred to here as afterhyperpolarization (AHP) (Figs. 1 and 3B). In contrast, there is a comparatively weak and more transient depolarizing after-reaction following ND motion. An attenuation of the early component of the test response is only detectable if the test response occurs while the after-reaction is still present (compare Fig. 3, A and B). Likewise, the small adaptation amplitude and its fast recovery during motion in the ND is paralleled by a low amplitude and a fast recovery time course of the after-reaction following ND motion (see Fig. 3).

These results suggest, that direction-selective adaptation is based on a postsynaptic cellular mechanism in the TCs, for example an increased K+ conductance or a decreased nonselective cation conductance, which on the one hand leads to a reduction in response strength during prolonged motion stimulation and on the other hand results in an AHP on stimulus cessation.

Correlation of AHP with dendritic Ca2+ accumulation

Motion stimulation in the PD has previously been shown to increase the Ca2+ concentration in TCs (Borst and Egelhaaf 1992; Dürr and Egelhaaf 1999; Egelhaaf and Borst 1995; Egelhaaf et al. 1993; Single and Borst 1998). By optical recording of fluorescence changes of the Ca2+ indicator fura-2 in the dendrites of HS and CH cells, we measured the relative changes in the intracellular Ca2+ concentration (Delta [Ca2+]i) in vivo during visual stimulation. HS cells showed significant rises in [Ca2+]i in response to adapting and test stimuli moving in the PD but almost no changes for ND motion (Fig. 2). As was shown previously, Delta [Ca2+]i differs from Delta EM in its time course: The slope of Delta [Ca2+]i is less steep than that of Delta EM and continues to rise when the latter already reaches a steady-state plateau (Dürr and Egelhaaf 1999). Furthermore, whereas the decay of Delta EM after the stimulus stops takes place within tens or hundreds of milliseconds, the decay of [Ca2+]i is characterized by time constants of several seconds.

To test a potential role of calcium in motion adaptation, we analyzed whether the strength of the AHP correlates with Delta [Ca2+]i in the dendrites of HS and CH cells. In response to PD motion stimuli of increasing duration, both cell classes show dendritic Delta [Ca2+]i with increasing peak amplitudes (Fig. 4A). The time course of both the increase in [Ca2+]i during motion stimulation and the decrease of [Ca2+]i after termination of stimulus motion is faster in CH than in HS cells as has been described earlier (Dürr and Egelhaaf 1999). During 4 s of ND motion (Fig. 4A, bottom), little deviation from baseline is seen in the mean Delta [Ca2+]i. A systematic investigation revealed that CH but not HS cells show, on average, decrements in dendritic [Ca2+]i in response to ND motion (unpublished results).



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Fig. 4. Relationship of [Ca2+]i and after-reaction of the membrane potential in response to motion of variable duration. Left: data for HS cells; right: data for CH cells. Photomontages constructed of raw fluorescence images of a southern cell of the horizontal system (HSS) and a dorsal centrifugal horizontal (DCH) cell, recording electrode visible in the left part of the picture, shown on top left and right, respectively; scale bars 100 µm. A: averaged Delta EM and Delta [Ca2+]i in response to visual motion stimuli of variable duration. Horizontal bars indicate period of stimulus motion (top to bottom: 200 ms, 500 ms, 1 s, 2 s, 4 s in PD and 4 s in ND). Time course of Delta EM (left part of each block) with shaded box, indicating time window for the calculation of the after-reaction (200 ms -2 s after stimulus cessation). Time course of Delta F/F (right part of each block) with shaded box, indicating single frame from which Delta F/F at the end of the motion stimulus was calculated. B: After-reaction of the membrane potential (Delta EAR) as a function of stimulus duration (plotted on a logarithmic scale). Mean values for stimulus motion in PD () and in ND (open circle ). C: Delta EAR following PD motion of variable duration (given beside each data point) as a function of Delta [Ca2+]i. Note the different scaling of the ordinate for HS- and CH-cells. Sample sizes: Electrophysiological data: HS-cells, n = 15-16 for PD and n = 7-15 for ND; CH-cells, n = 7-8 for PD and n = 5-6 for ND. Data on Ca2+: HS-cells, n = 5; CH-cells, n = 4. Error bars denote SE in all cases.

After-reactions of the membrane potential were quantified in a time window of 1.8 s, starting 0.2 s after the stimulus stopped. An AHP occurs only when PD motion lasts for at least 0.5 s. If a shorter stimulus is presented, the depolarization decays rather slowly and the after-reaction is on a depolarized level (Fig. 4A). Plotting the amplitude of the after-reaction (Delta EAR) to PD motion versus the logarithm of stimulus duration reveals that for both cell classes Delta EAR steadily decreases with increasing stimulus duration (Fig. 4B). In contrast, for ND motion, HS cells show small after-depolarizations, the amplitudes of which do not depend on stimulus duration. Although CH cells show hyperpolarizing after-reactions to ND motion, again, there is no monotonic dependency of Delta EAR on stimulus duration.

Finally, there is a monotonic relationship between AHP and Ca2+ accumulation for PD motion both for HS and CH cells. This is demonstrated by plotting Delta EAR as a function of the relative fluorescence change Delta F/F at the end of the motion stimulus (Fig. 4C).

Dependence on stimulus velocity of Ca2+ accumulation and motion adaptation

The response of visual motion-sensitive neurons such as HS and CH cells is not only determined by the duration of the stimulus but also by other stimulus parameters, such as velocity. Therefore we investigated whether a correlation between AHP and [Ca2+]i also exists if the stimulus strength is altered by varying the velocity of PD motion. Due to the nature of motion detection, HS and CH cells show membrane-potential responses of maximal size at a certain temporal frequency, below and beyond which the amplitudes decline (Dürr 1998; Egelhaaf and Borst 1989; Hausen 1982b). In HS cells, the corresponding velocity tuning of Delta [Ca2+]i is known to follow a similar characteristic (Dürr 1998). In contrast, CH cells exhibit a discrepancy between the velocity tuning of the [Ca2+]i response and that of the electrical response (Dürr 1998). In a plot of Delta EAR values versus Delta F/F, both determined in the same way as for Fig. 4C, but for motion stimuli of constant duration varying in temporal frequency, a monotonic relationship between the two parameters is obtained for HS but not for CH cells (Fig. 5).



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Fig. 5. Relationship of Delta [Ca2+]i and Delta EAR in response to PD motion of variable temporal frequency. Determination of Delta F/F and Delta EAR as in Fig. 4. Sample sizes: electrophysiological data: HS cells, n = 30; CH cells, n = 27-31. Data on Ca2+: HS cells, n = 9-12; CH cells, n = 10-12. Error bars denote SE.

At very high velocities (24-Hz temporal frequency), HS cells respond with a distinct, though transient, membrane depolarization, which is accompanied by little or no [Ca2+]i increases (Dürr 1998). As expected if the AHP depends on cytosolic Ca2+, membrane potential changes induced by 24-Hz stimuli are not followed by an AHP (Fig. 6A). In contrast, an after-depolarization follows, comparable in strength and time course to the one seen after ND motion.



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Fig. 6. Adaptation caused by stimuli moving at a high velocity. A: averaged Delta EM recorded from HS cells (n = 10) when a stimulus with a temporal frequency of 24 Hz and a duration of 2 s is presented either in PD (top) or ND (bottom). B: Delta EM (---) and Delta [Ca2+]i (···) on presentation of a reference 2-Hz stimulus, followed after 1.9 s by a 24-Hz adapting stimulus and after an interval of 100 ms by a 2-Hz test stimulus. Averaged time course recorded from HS cells (electrophysiological data: n = 5; data on Ca2+: n = 4 for PD and n = 3 for ND).

Although adaptation renders membrane potential responses more transient, the time course by itself cannot serve as an unequivocal indicator of adaptation because, inherent to the mechanism of motion detection, responses are more transient for fast stimuli than for slow stimuli (cf. Figs. 1 and 6A) (Egelhaaf and Borst 1989). However, differences in the time course of responses to PD and ND motion may indicate direction-selective adaptation. The responses to fast moving stimuli shown in Fig. 6A exhibit two direction-selective characteristics: first, the response to ND motion remains on a slightly larger steady-state membrane potential level than the PD response. Second, the after-reactions of responses to PD and ND motion are not mirror symmetrical, particularly because responses to both PD and ND motion are followed by after-depolarizations. Since stimuli moving at a temporal frequency of 24 Hz were shown to cause at most very slight Delta [Ca2+]i, Ca2+-dependent adaptation cannot be assumed to be the sole determinant of direction-selective effects.

Direction-selective adaptation potentially independent of Ca2+ accumulation

As outlined in the preceding paragraph, fast moving stimuli cause very transient membrane potential responses. During PD motion, these are accompanied by at most very small [Ca2+]i increases (Dürr 1998). To assess the strength and direction selectivity of motion adaptation caused by a fast moving stimulus, hence without pronounced concurrent Ca2+ increase, HS cells were stimulated by the following protocol: First, 2-Hz motion was presented for 2 s as a reference stimulus. After 1.9 s, 24-Hz motion was presented for 2 s as the adapting stimulus, followed after 100 ms by a 2-Hz test stimulus (Fig. 6B). During PD motion, the transient response to the test stimulus is attenuated relative to that elicited by the reference stimulus. The size of this effect renders it unlikely that it is completely due to the initial 2-Hz stimulus because adaptation elicited by such a stimulus has recovered after an interval of 4 s (see Fig. 3A). Thus the attenuation is at least partially due to the 24-Hz stimulus. The adapting effect of 24-Hz motion is probably direction selective because during ND motion, the transient response to a test stimulus is even slightly enhanced in 9 of 13 recordings if a 24-Hz stimulus is presented first (Fig. 6B). The time course of Delta [Ca2+]i during 24-Hz motion (Fig. 6B, · · · ) is consistent with the finding that fast moving stimuli have only little influence on [Ca2+]i (Dürr 1998). Nevertheless [Ca2+]i increases that are hardly detectable by measuring Delta [Ca2+]i across the entire dendrite could be fairly large in close proximity to the cell membrane. Because such [Ca2+]i increases could not be detected with our available imaging techniques, it appears at present most plausible to conclude that fast moving stimuli cause direction-selective adaptation by a mechanism that is probably independent of [Ca2+]i increases.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Prolonged visual motion stimulation reduces the amplitude of a response to subsequent stimuli in directionally selective neurons of the fly. The effect, although present in both directions of motion, is much more pronounced and longer lasting for motion in the PD of the neuron. This direction selectivity is also observed in the after-reactions: an AHP, increasing in size with stimulus duration, is built up by PD motion, whereas comparable after-depolarizations are not elicited by ND motion. Adaptation and AHP could be based on the same physiological mechanism, for instance, a reduced excitatory or an increased inhibitory conductance. We propose dendritic Ca2+ as a physiological mediator of such a process. In our approach, direction selectivity of motion adaptation helps to discriminate mechanisms that are potentially based on Ca2+ from Ca2+-independent ones.

Role of Ca2+ for the modulation of responsiveness

When stimulus duration is varied, AHP and dendritic Delta [Ca2+]i are correlated in HS and CH cells (Fig. 4C). Although there are several possible mechanisms to cause an AHP, Ca2+ seems a likely candidate, given the evidence from other preparations. AHPs were shown to be mediated by Ca2+-dependent K+ currents in a multitude of cell types (review: Sah 1996). This physiological mechanism is responsible for spike frequency adaptation in neurons (e.g., Chitwood and Jaffe 1998; Lo et al. 1998). Similarly, sensitivity changes in sensory cells are often mediated by ion channels that are directly or indirectly regulated by Ca2+ (reviews: Fettiplace and Fuchs 1999; Koutalos and Yau 1996; Menini 1999). For example, in vertebrate photoreceptors Ca2+ regulates sensitivity by both decreasing the affinity of the cGMP-gated cation channel for cGMP and inhibiting guanylate cyclase.

However, the correlation of the AHP and Delta [Ca2+]i observed in TCs does not prove that Ca2+ controls the AHP. To provide unequivocal evidence, it would be necessary to interfere effectively with [Ca2+]i in TCs. Single (1998) reported an increase in the frequency of spike-like discharges superimposed on graded membrane potential responses by iontophoretic injection of the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) into the cytoplasm of TCs. This hints at a role of Ca2+ in the modulation of cellular excitability. In our own experiments, injection of BAPTA into single HS cells caused no significant change in AHP and adaptation. It has to be noted, however, that in vivo recording of TCs cannot yet be done with patch electrodes. Thus intracellular buffer concentrations cannot be controlled; something that would be necessary to rule out the existence of a BAPTA-blockable, thus Ca2+-dependent, mechanism in motion adaptation.

Possible Ca2+-independent mechanisms leading to an AHP are for example slowly inactivating K+ conductances, the Na+/K+ pump (e.g., Gerster et al. 1997; Jansen and Nicholls 1973; Jansonius 1990) or Na+-dependent K+ channels (e.g., Kim and McCormick 1998; Schwindt et al. 1989), the latter of which have been concluded to exist in blowfly HS neurons (Haag et al. 1997). Measuring the conductance change following motion stimulation and comparing its time course with that the AHP and of Delta [Ca2+]i could help to determine the physiological mechanism underlying adaptation. However, due to ion channel kinetics and to putative differences in bulk cytosolic and near-membrane Ca2+ dynamics, the time course of a Delta [Ca2+]i is not necessarily identical to that of a Ca2+-dependent conductance (Jahromi et al. 1999; Sah and Clements 1999). Furthermore because different physiological mechanisms could cause similar conductance changes, an unequivocal identification of the conductance leading to AHP would additionally require pharmacological studies, which are beyond the scope of this study.

Comparison of HS and CH cells

In HS cells, the AHP and dendritic Delta [Ca2+]i are correlated, regardless of whether stimulus strength is altered by varying the duration or the temporal frequency of motion. CH cells, on the other hand, exhibit a monotonic relationship between AHP and Delta [Ca2+]i only for increases in stimulus duration but not for changes in temporal frequency (Figs. 4C and 5). This is reminiscent of the finding that the temporal frequency tuning of electrical and Ca2+ responses is similar in HS cells but different in CH cells (Dürr 1998). Clearly, [Ca2+]i is not the only determinant of the AHP in CH cells because AHPs were also present following ND stimuli (Fig. 4B). Further differences in motion-induced Delta [Ca2+]i between HS and CH cells, as found with respect to the time course and the spatial spread of Ca2+ accumulation, and the efficacy of ND stimuli in eliciting Ca2+ decrements have been found (Dürr 1998; Dürr and Egelhaaf 1999). At present it is unclear to what extent these differences correspond to cell-class-specific properties of motion adaptation, e.g., with respect to its temporal and spatial characteristics. So far, the differences between cell classes have been discussed to reflect a purely modulatory role of Ca2+ in HS cells and a putative involvement in transmitter release at the dendrodendritic synapses of CH cells.

Implications for concepts of motion adaptation in TCs

In the past, only presynaptic changes have been discussed to cause motion adaptation in TCs of the fly. One possible mechanism, although controversially discussed, is a decrease of EMD delay time constants, which would shift the temporal frequency tuning of TCs to higher velocities (de Ruyter van Steveninck et al. 1986; but see also Harris et al. 1999a). Borst and Egelhaaf (1987) showed that adaptation cannot only be elicited by motion stimulation but also by temporal modulation of luminance (flicker). However, the fact that motion stimuli were more effective than flicker stimuli in causing motion adaptation can be interpreted as a first hint that more than a single mechanism underlies motion adaptation in TCs. Recently Harris et al. (1999b) provided evidence for a reduction of contrast gain in HS cells induced by presenting adapting motion in any direction, including orthogonal motion, but not by presenting flicker. The most plausible explanation for this observation is an adaptation mechanism located presynaptic to TCs.

Here we suggest a postsynaptic mechanism for motion adaptation. We show that motion adaptation is to a certain extent associated specifically with PD motion. The TC itself is a likely location for the physiological mechanisms associated with direction-selective adaptation because it is regarded as the stage of motion processing where full direction selectivity is computed from only weakly direction-selective inputs (Borst and Egelhaaf 1990; Borst et al. 1995; Egelhaaf et al. 1989; Single et al. 1997). However, direction-selective motion adaptation is not simply determined by the excitation of the TC because membrane potential responses exhibit much faster time courses compared with the buildup of adaptation and recovery from it. Rather, temporally integrating the membrane potential response would be necessary to predict the strength of adaptation. Due to the time course of its accumulation, Ca2+ could mediate such a computational function. Nevertheless direction-selective motion adaptation seems not to be caused exclusively by a Ca2+-dependent mechanism. Fast-moving stimuli are shown to produce adaptation in a direction-selective manner without leading to considerable Ca2+ accumulation, thus providing further evidence for a heterogeneous origin of motion adaptation.

Comparison with motion-sensitive neurons in other species

Motion adaptation and its underlying mechanisms have also been investigated in motion-sensitive neurons of other species. Additionally, psychophysical studies (e.g., Clifford and Langley 1996b; Clifford and Wenderoth 1999) and functional magnetic resonance imaging (Tootell et al. 1995) have been performed on man to elucidate the nature of motion adaptation and its connection with the well-studied motion aftereffect. In this psychophysical phenomenon, prolonged viewing of a pattern moving in one direction results in illusory perception of movement in the opposite direction after the stimulus stops (review: Sekuler et al. 1978). In a direction-selective neuron such as a HS or CH cell, ND motion is signaled by membrane hyperpolarization. An AHP following prolonged PD motion might be "misinterpreted" by a subsequent processing stage as ND motion and could thus serve as a physiological explanation of the motion aftereffect.

The first physiological study on motion adaptation was by Barlow and Hill (1963) in the retinae of rabbits. They found that direction-selective ganglion cells exhibit a drop of discharge frequency below the spontaneous rate when movement stops after a long-lasting stimulation period.

Maddess et al. (1991) report that adaptation induces a shift of the velocity response curve toward higher velocities in motion-sensitive neurons of the butterfly Papilio aegeus. They propose the idea of piece-wise linearity, i.e., neurons responding linearly to changes in stimulus strength within each adaptation range. Similar changes in the temporal frequency tuning were reported for directional neurons in the nucleus of the optic tract (NOT) of the wallaby Macropus eugenii (Ibbotson et al. 1998). Adaptation, as assessed by determining decay time constants of responses to step-wise grating displacements (impulsive stimuli), was pronounced for PD motion but only weak with adapting stimuli moving in the ND or modulating temporally in luminance.

In the visual cortex of cats (area 17), adaptation also altered the temporal frequency tuning of motion-sensitive neurons, but instead of shifting the response curves, the strongest aftereffect occurred at the adapting frequency, with smaller effects both above and below it (Saul and Cynader 1989). This result is difficult to reconcile with a model incorporating adaptation-induced changes of time constants in EMDs unless there exist several kinds of EMDs with different delay filters. Also in cat visual cortex, Giaschi et al. (1993) report that in simple cells two processes are evident in the time courses of both the decrement of responsiveness during PD adaptation and its subsequent recovery: a rapid direction-independent and an additional slow, direction-selective process. This is very similar to the results on motion adaptation in fly TCs as described in the present study. Several further studies also show direction selectivity of motion adaptation in cat visual cortex (e.g., Hammond et al. 1988; Marlin et al. 1988) and area MT of the owl monkey (Petersen et al. 1985). However, the meaning of direction selectivity of adaptation in their and in our study is not completely congruent because most of the cortical neurons investigated are not fully direction selective in a strict sense. When stimulated with ND motion, the cortical neurons show weak response increments or no responses rather than decrements below resting activity as is found in fly TCs. Along with the different response characteristics of motion-sensitive cells, there probably exists a variety of mechanisms mediating motion adaptation. The present study presents evidence for a postsynaptic mechanism, namely dendritic Ca2+ accumulation, which can account for several features of motion adaptation in TCs of the fly, such as its direction selectivity, time course, and local restriction.


    ACKNOWLEDGMENTS

We thank B. Kimmerle for useful discussions of this work and R. Feiler for support in the programming of the A/D converter. The constructive comments of two anonymous referees are gratefully acknowledged.

This work was supported by the Deutsche Forschungsgemeinschaft.

Present address of V. Dürr: Abteilung für biologische Kybernetik und theoretische Biologie, Fakultät für Biologie, Universität Bielefeld, Postfach 10 01 31, D-33501 Bielefeld, Germany.


    FOOTNOTES

Address for reprint requests: R. Kurtz, Lehrstuhl für Neurobiologie, Fakultät für Biologie, Universität Bielefeld, Postfach 10 01 31, D-33501 Bielefeld, Germany (E-mail: rafael.kurtz{at}biologie.uni-bielefeld.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 January 2000; accepted in final form 6 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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