Directionality and Inhibition in Crayfish Tangential Cells

Raymon M. Glantz

Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Glantz, Raymon M. Directionality and inhibition in crayfish tangential cells. J. Neurophysiol. 79: 1157-1166, 1998. The purpose of this study was to characterize the inhibitory mechanism(s) associated with directionally selective motion detection (DS) in nonspiking tangential cells of crayfish optic lobe. The experiments employed intracellular recording of synaptic potentials elicited with sinewave gratings and pharmacological techniques. Previous studies established that tangential cells are subject to bicuculline-sensitive GABA-mediated inhibition. In this study DS was reduced by 90% by bicuculline. The reduction in DS was accompanied by a substantial increase in the response to null-direction motion. Bicuculline also altered the response to pulses of illumination. The magnitude and time course of inhibition were derived from the time varying difference between the control light response and that elicited during bicuculline perfusion. Both the inhibitory delay (relative to excitation) and the inhibitory amplitude are close to the expectations of a linear model of DS. The inhibition is not prolonged with respect to excitation but its risetime is ~2.5 times longer. The result implies a longer time constant in the inhibitory pathway relative to that in the excitatory pathway and places limits on the frequency response of inhibition and DS. The velocity-dependence of DS is related to the time course of inhibition. The stimulus drift velocity eliciting maximum directionality is inversely proportional to the inhibitory delay. Bicuculline did not influence orientation selectivity. It is concluded that the quantitative features of bicuculline-sensitive, GABA-mediated inhibition are consistent with a linear model of DS.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Directionally selective motion detection is an important example of a spatiotemporal computation implemented by a network of neurons. The unique feature of most directional systems is the inhibition of the response to motion in the antipreferred (null) direction (Barlow and Levick 1965; Ganz and Felder 1984; Jagadeesh et al. 1993). Theoretical and simulation studies (Bouzerdoum and Pinter 1991; Glantz 1994; Grzywacz and Koch 1987; Reid et al. 1991) indicate that the inhibitory component of the motion detector's receptive field must be temporally and spatially asymmetric relative to the excitatory subfield.

Pharmacological methods have been employed in efforts to further characterize the inhibitory mechanism(s) in directional selectivity (DS). In rabbit and turtle retina (Ariel and Adolph 1985; Caldwell et al. 1978), cat visual cortex (Sillito 1975), and fly optic lobe (Schmid and Bülthoff 1988), the early studies indicated that gamma -aminobutyric acid (GABA) antagonists abolish DS. With one exception (Nelson et al. 1994), more recent studies (Brotz and Borst 1996; Cohen and Miller 1995; Egelhaaf et al. 1990; Kittila and Massey 1997) confirm the earlier result but challenge some of the initial interpretations and also imply considerable complexity in underlying DS mechanisms.

Recent studies of nonspiking interneurons (tangential cells) in crayfish optic lobe (Glantz 1994; Glantz and Bartels 1993) have provided evidence for a simple DS mechanism based upon the linear (or nearly linear) interaction of excitation and delayed inhibition arising from asymmetric receptive subfields. Both the experimental and theoretical studies relied, in part, upon the results of prior qualitative studies (Pfeiffer-Linn and Glantz 1989), which indicated that tangential cells are subject to bicuculline-sensitive GABA-mediated inhibition. It has not been established in this system that GABA antagonists block DS nor is it known if the GABA mediated inhibition has the temporal properties required by the linear model of DS (Glantz 1994). This study was undertaken to address these issues.

An additional aspect of movement detection is that the phenomenon often appears to be coupled with selectivity for one or more other stimulus features. A notable example is that the motion axis for DS in visual cortical neurons is typically at 90° to the optimal orientation axis (Hubel and Wiesel 1959). In rabbit retina and in a subset of fly lobula neurons, DS appears to be linked to a selectivity for the stimulus length (Caldwell et al. 1978; Egelhaaf et al. 1993; Warzecha et al. 1993). In other cells, DS is expressed over a defined range of stimulus temporal frequencies (e.g., fly) (Eckert 1980; Egelhaaf et al. 1989) or velocities (Baker 1988; Baker et al. 1991; Glantz 1994). If the coupling between DS and other stimulus features is obligatory, the joint selectivity may have implications for the DS mechanism. In cat cortical neurons, DS and orientation selectivity can be dissociated by bicuculline (Nelson 1991) but in rabbit retina, DS and size selectivity diminish in parallel after picrotoxin treatment (Caldwell et al. 1978). In this study the effects of bicuculline on DS and orientation selectivity are compared and the velocity-dependence of DS is related to the time course of inhibition.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

The experimental procedures for visual stimulation and recording were the same as those in Glantz (1994). Adult crayfish of both sexes and 10-12 cm in length were exsanguinated at 8°C by exchanging oxygenated and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered crayfish saline for hemolymph over a 1-h period. The chelipeds and rostrum were removed and the eyestalks were glued in their sockets with cyanoacrylate adhesive.

The animals were clamped in a specially constructed plexiglass chamber filled with oxygenated saline. The cuticle and sinus tissue were excised from the dorsal eyestalk such that the major landmarks of the optic lobe (lamina, medulla externa, etc.) could be visualized. After the dissection, the level of the bath was lowered to just below the eyestalk. The optic lobe remained under fluid however because of a wicking action of the eyestalk tissues.


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FIG. 1. Time course of inhibition is derived from difference between control light response and that elicited during bicuculline perfusion. Light pulse, indicated by bar on x-axis, was 0.7 s long and log intensity was -0.5. D, inhibitory delay.


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FIG. 2. A: scatterplot of directional selectivity index (DSI) versus normalized repolarization. (), least squares regression with a slope of 0.44 DSI units per 100% repolarization for n = 51 cells. Product-moment correlation coefficient is 0.56. B: scatterplot of stimulus temporal frequency for maximum DSI versus width of peak hyperpolarization at half-maximum amplitude. Spatial wavelength was 30°/cycle. Linear regression for temporal frequency versus inverse of width at half-maximum yielded a slope of 0.076 Hz/s-1 and a correlation coefficient of 0.78, n = 32.

Electrophysiological recordings were made with micropipettes filled with potassium acetate and of 100-200 MOmega resistance. The signals were led to an Axoclamp IB amplifier and then to a storage oscilloscope and tape recorder. Tangential (Tan1) neurons were impaled in the distal medulla and adjacent cell body area and identified by functional criteria established in Wang-Bennett and Glantz (1987) and extended in Glantz (1996). These criteria include the impalement site, the time course of the hyperpolarizing responses to steps of illumination, and the dimensions of the receptive field. Bicuculline perfusion was performed with a two-channel pressure injection system that delivered 100 µM bicuculline methiodide or saline through glass pipettes with 5.0-µm tip diameters. The perfusion rate was 5.0 µl/min. The pipette tips were placed on top of the external chiasm, 100-200 µm distal to the medulla externa. The saline was used to wash the eyestalk after bicuculline perfusion.

The stimulus system was described in Glantz and Bartels (1994). It consisted of two pathways that can be alternated by movement of a single mirror. One pathway consisted of a 7-mW He-Neon laser, an electromagnetic shutter, and neutral density wedge. The spot size was adjusted to 5.0 mm diam (24°) with convex lenses and the stimulus position was controlled by two mirrors mounted on galvanometers. The maximum intensity measured at the eye was 0.1 mW/mm2. The stimulus was delivered to the eye via a fiber optic lens (described in Brodie et al. 1978), which is built into the wall of the recording chamber. The lens converts the two-dimensional image on a 2.5-cm flat surface to hemispherical coordinates spanning 120°. The inner hemispherical surface functions as a translucent, rear-projection screen. The geometric center of the cornea of the eye was placed at the optical center of the curved lens surface.

The second optical pathway produced sinewave gratings that were focused on the flat surface of the fiber optic lens. The gratings were produced on the CRT of a Hitachi display oscilloscope driven by a Picasso Image Synthesizer (Innesfree, Cambridgeshire, UK) under the control of a PC computer. The data were digitized (off-line) with an A/D converter and PC computer. The sampling rate was 400 Hz. Every stimulus condition (described below) was repeated 5-40 times according to the experiment. During data analysis baseline drift was removed with a detrending program and the responses were averaged with programs written in MATLAB (Natick, MA).


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FIG. 3. Time course of bicuculline action on directional selectivity. Four panels show averaged preferred- () and null-direction (- - -) responses in control conditions and at indicated durations of bicuculline perfusion. DSI declined from 0.46 to 0.04 in 280 s. Each trace is average of 10 response cycles. Remporal frequency is 1.0 Hz and spatial wavelength is 30°/cycle.


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FIG. 4. Directional selectivity is reversibly abolished by bicuculline. A: control responses. () and (- - -) are preferred- and null-direction responses, respectively. DSI is 0.31. B: during bicuculline perfusion, DSI is 0.04. C: after a 15-min wash, DSI is 0.33. Temporal frequency is 3.6 Hz and spatial wavelength is 30°/cycle. D: comparison of response amplitudes in control (C), bicuculline perfusion (B), and wash (W).

Tangential cells respond to a drifting sinewave grating with a phase-sensitive oscillation in membrane potential. Directional and orientation selectivity were assessed from the direction-dependent variations in the amplitude of the oscillatory response. Responses were elicited for eight directions of drift spaced 45° apart. The stimulus orientation was always 90° to the drift direction. Each sample consisted of responses to 20-40 stimulus cycles depending upon the signal to noise ratio. To control for nonstationarities, half of each sample was obtained between successive clockwise rotations and the other half between successive counter-clockwise rotations. Response magnitudes were determined from the averaged response.

The results are based upon changes in the response after bicuculline perfusion for three sets of measurements; directional selectivity, orientation tuning, and the incremental response to various light pulse intensities. For each measurement, the responses elicited in the presence of bicuculline were compared with the control responses of the same neuron.

Directional selectivity was measured from the difference in responses to gratings drifting in the preferred and null directions, which are 180° apart. The preferred direction elicited the largest response amplitude among the eight directions tested. The data are evaluated by the directional selectivity index (DSI)
DSI = (<IT>P − N</IT>)/(<IT>P + N</IT>) (1)
where P and N are the response amplitudes in the preferred and null directions respectively.


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FIG. 5. Bar plot of average normalized response amplitudes in control and during bicuculline perfusion for 12 cells. Error bars are means ± SD. Responses for each cell were normalized to amplitude of preferred-direction response in control. P, preferred direction; N, null direction.


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FIG. 6. Bicuculline does not influence orientation selectivity while abolishing directionality. Top: responses to preferred- and null-direction drift. Preferred direction was 315° (). Bottom: responses at ±90° to preferred direction. Each trace is average of 10 cycles. Temporal frequency was 0.5 Hz, spatial wavelength was 30°.

Orientation selectivity was assessed from the difference between responses elicited by preferred-direction motion and mean of the responses elicited by motion at ±90° to the preferred direction (A). These data were evaluated with the orientation selectivity index (OSI)
OSI = (<IT>P − A</IT>)/(<IT>P + A</IT>) (2)
Step responses were elicited in a stimulus regime which elicits constant magnitude responses for separate presentations of the same stimulus intensity. The receptive field was located by horizontal and vertical scans of the laser beam through the visual field and the beam was centered on the region of maximum response. After 5 min of adaptation to a low level (10-7 mW/mm2) of illumination, pulses of 0.5-0.7 s duration were presented at 8-s intervals. Stimulus intensity was varied from threshold to saturation (typically a 2.5-3 log10 intensity range) in 0.5 log unit steps. Each intensity was repeated five times and the five responses were averaged to determine the intensity-dependent waveform of the postsynaptic potential (PSP).

The influence of bicuculline on the step response was determined from the response waveform as shown in Fig. 1. The Tan1 control (C) step-response consists of a transient peak hyperpolarization and subsequent partial repolarization to a plateau phase. The repolarization may culminate in a distinct trough (as in Fig. 1). The magnitude of the repolarization is the difference between the peak amplitude and the minimum of the plateau phase. For comparisons between cells, the repolarization magnitude is normalized to the amplitude of the peak transient. During bicuculline perfusion (B) (as in Fig. 1, broken trace) the plateau phase is typically increased relative to control. The time course of bicuculline-sensitive inhibition (I) is determined from the difference function: I(t) =C(t- B(t), indicated in the solid depolarizing trace in Fig. 1. For comparisons between excitation and inhibition, the difference function is taken as the inhibitory response and the response elicited in the presence of bicuculline (B) is taken as the excitatory event. The latencies for excitation, E(tl), and inhibition I(tl), are the times between stimulus onset and the achievement of 10% of the respective maximum responses (D in Fig. 1). The inhibitory delay (D) is: D = I(tl- E(tl).

The ratio of inhibition to excitation (I/E) determines the upper limit of DSI (Glantz 1994). The relative magnitude of inhibition, I/E, is the maximum ratio of the two responses evaluated at the same poststimulus time: I/E = max [I(t)/E(t)].


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FIG. 7. Polar plot of response amplitudes for 8 directions of motion in control () and during bicuculline perfusion (- - -). Note that stimulus orientation is 90° to direction of motion.


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FIG. 8. Averaged responses of 10 neurons, each normalized to response for control, preferred direction of motion. P is preferred-direction response; P - 90 is average amplitude of responses at ±90° to preferred direction. Error bars are ± 1.0 SD.

To establish the time course of bicuculline action, pulse or DS measurements were carried out during the perfusion. For DS, the preferred- and null-directions were alternately tested just before and during bicuculline perfusion. Alternatively, the step response was elicited at 8-s intervals. The stimulus regime was repeated in control conditions until a steady-state response was evident in the superposed traces of a storage oscilloscope.

The results are derived from 18 neurons, which were tested for DS, subjected to bicuculline perfusion and further analyzed for the time course of inhibition. For one analysis, results were compiled from the control data in this study and data obtained with the same procedures in a previous study (Glantz 1994). One analysis is based upon response risetime, which is the interval between 10 and 90% of maximum response.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

In the proposed model of DS (Glantz 1994), the maximum ratio of the preferred-to null-direction response is a linear function of the ratio of inhibition (I) to excitation (E) or I/E. Because stronger inhibition produces a deeper repolarization of the response to a pulse of light (i.e., a smaller plateau phase) there should be a quantitative relationship between the magnitude of the repolarization and the strength of directionality. In 51 cells examined under control conditions, DSI varied between cells from 0.08 to 0.83 and the repolarization of the pulse response varied from 4 to 100% of the peak amplitude. Figure 2A is a scatter plot of DSI versus the normalized repolarization for these 51 Tan1 neurons. The slope of the least squares regression is 0.44 DSI units per 100% repolarization and the product-moment correlation coefficient is 0.56 (P < 0.01). The latter implies that variations in the repolarization magnitude are indicative of factors that contribute to DS. The shallow slope of the regression however and the modest size of the correlation coefficient imply that the repolarization may reflect variables other than inhibition, such as the time course of the presynaptic response.

In the linear DS model the drift velocity for maximum directional selectivity (Vmax) is equal to the spatial sampling interval, p, divided by the inhibitory delay (d); Vmax = p/d. Because the width of the transient peak of the pulse response should be proportional to the inhibitory delay, it follows that Vmax should be inversely proportional to width of the peak hyperpolarization. In Fig. 2B the temporal frequency (F) associated with Vmax is plotted against the width of the transient response at half-maximum amplitude. The spatial wavelength was 30°/cycle and Vmax = 30° × F. The linear regression between Vmax and the inverse of the width of the peak at half maximum exhibited a correlation coefficient of 0.61 for 32 measurements (P < 0.01). The narrowest transients (25 ms wide) are associated with a Vmax of 120°/s, while the broadest transients are 10-fold wider and associated with a Vmax of 6-15°/s. Thus features of the Tan1 response to a flash are correlated with the magnitude of DSI and the velocity-dependence of directionality. These correlations presumably reflect the magnitude and speed of inhibition.

Because bicuculline blocks the action of GABA and partially blocks repolarization, it should also block DS. The effect of bicuculline on DS was generally observable in 1 min and the action complete in 2-5 min, as in Fig. 3. In this experiment, the control DSI was 0.46. After 60 s of bicuculline perfusion the DSI was reduced to 0.21 and after 280 s the DSI was 0.04. Bicuculline had very little effect on the magnitude of the preferred-direction response but it increased the null-direction response by 50 to 100% as in Fig. 3.

Figure 4 shows the results from another cell in which the action of bicuculline on DS was reversed after a 15-min saline wash. The control null-direction response is 0.7 mV as in Fig. 4A. Bicuculline doubles the size of the null-direction response and reduces DSI from 0.31 to 0.04 (as in Fig. 4B), with only a small effects on the preferred-direction response. After the wash DSI and the null-direction response are restored as in Fig. 4C. Figure 4D compares the response amplitudes in the three conditions. Bicuculline action was reversed in three experiments. One cell did not reverse in 30 min and reversibility was not tested in another eight cells, in which either the penetration was lost or visual sensitivity declined before a wash could be initiated.


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FIG. 9. Time course of bicuculline action on light pulse response. Light pulses of 0.7 s duration and log intensity -0.5 were presented at 8-s intervals. Each trace is average of 6 successive responses. Duration of bicuculline perfusion (in seconds) at end of each sample is indicated above each trace. Scale is 1.0 s and 1.0 mV. Note increase in plateau phase amplitude between 96 and 144 s of perfusion.


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FIG. 10. Tangential cell responses to light pulses of varying intensity. A: control. B: after 5-10 min of bicuculline perfusion (BICUCULLINE). Difference functions are control traces minus responses in bicuculline. Pulse duration is 0.7 s and intensity steps are 0.5 log10 units between log intensity -3 and -0.50. Stimuli at 8-s intervals. Each trace is average of 5 responses.

Figure 5 summarizes the results for 12 directionally selective neurons. In the control condition, the null-direction response is less than half that in the preferred direction. Bicuculline diminishes the preferred-direction response by 11% and increases the null direction response by 69%. These changes reduce the DSI from 0.36 ± 0.05 (SD) to 0.04 ± 0.05 (SD) (P < 0.01).

Orientation selectivity

About 70% of all tangential cells examined exhibit at least a modest degree of orientation selectivity (OSI > 0.3). For these neurons the range of OSI is 0.3-0.8 and the mean OSI is 0.52 ± 0.14 (SD). The mean width of the tuning curve at half-maximum response is 42 ± 31° (SD). In contrast to its effects on DS, bicuculline had no effect on orientation tuning. Figure 6 shows responses to stimuli moving along the preferred-null axis (315-135°) and at ±90° to the preferred direction. The preferred-direction response was three times that in the orthogonal directions. After bicuculline perfusion sufficient to block DS, the OSI was 0.55 compared with the control OSI of 0.50. These results and those obtained for the four other motion directions are summarized in a polar plot in Fig. 7. Comparable results were obtained in 10 neurons and the averaged result is shown in Fig. 8. Neither the preferred-direction response, nor those in the orthogonal directions were significantly altered by bicuculline.

Inhibitory delay and magnitude

As noted above, there is qualitative evidence that bicuculline blocks GABA-elicited depolarization and increases the magnitude of the plateau phase of the light-elicited hyperpolarizing response. From these results it was inferred that a bicuculline-sensitive GABA receptor participates in the repolarization of Tan1.

In several experiments the pulse responses, elicited at 8 s intervals, were used to monitor the time course of bicuculline action. The five responses in Fig. 9 are the control response (t = 0) and the response at the indicated duration of bicuculline perfusion. By 144 s after the start of perfusion, the plateau phase exhibited a substantial increase in amplitude and the trough associated with repolarization was substantially diminished.

The influence of bicuculline is expressed over a wide range of stimulus intensities and the inferred inhibition exhibits only a modest degree of intensity-dependence. Figure 10A shows the variations in the Tan1 response as a function of intensity in control conditions and Fig. 10B (BICUCULLINE) shows the responses during bicuculline perfusion. Bicuculline produces a small (5-10%) enhancement of the transient hyperpolarization and a more substantial enhancement (30-40%) of the plateau response. Thus bicuculline diminishes the amount of repolarization. In Fig. 10A, at the highest intensities, the plateau phase amplitude of the control response is about half that of the peak amplitude. In the presence of bicuculline (as in Fig. 10B) the comparable ratio is 0.7.

If the system is linear (e.g., no voltage-sensitive conductances), the difference between the control response and that elicited in the presence of bicuculline should reflect the time course and magnitude of inhibition. Previous current-clamp studies (Pfeiffer and Glantz 1989) and more recent voltage-clamp studies (data not shown) reveal no evidence of a voltage-dependent conductance in the relevant region of membrane potential. The difference function I(t) is shown in Fig. 10B (DIFFERENCE). Except for a single response (at log intensity 2.5) the difference functions peak at about 150-ms poststimulus, with a maximum of 4.0-5.0 mV. The results in Fig. 10B show that the magnitude of inhibition and the time to peak inhibition are relatively insensitive to intensity. The essential result is displayed in Fig. 10C, which shows the three response waveforms [control (hyperpolarizing solid trace), bicuculline (broken line) and the difference function (depolarizing solid trace)] at a single intensity(log I = -1.0). Note that the difference function is delayed in time relative to the excitatory function and that the maximum amplitude is ~5.0 mV.

Because the time course and magnitude of inhibition are determinants of directionality, it is useful to consider their intensity-dependence. Figure 11 shows the effect of bicuculline as a function of pulse intensity for the salient features of the response. Bicuculline (- - -) increases both the peak transient and plateau amplitudes (as in Fig. 11, A and B, respectively). The enhancement of the peak transient in bicuculline indicates that inhibition commences during the rising phase of the hyperpolarizing response. Figure 11B (open circle ) represents the magnitude of the difference function that saturates in parallel with the plateau response. The ratio of inhibition to excitation, I/E, declines from 0.5 to 0.3 as the light intensity increases (Fig. 11C).


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FIG. 11. Intensity-dependence of tangential cell excitatory and inhibitory responses. A: amplitude of peak transient hyperpolarization in control condition () and in presence of bicuculline (- - -). B: amplitude of plateau phase response in control conditions() and during bicuculline perfusion(- - -). C: intensity-dependence of percent inhibition, i.e., I/E. D: Intensity-dependence of inhibitory delay.

Because the neuron described in Fig. 11 exhibited a DSI of 0.31, an I/E of 0.47 was expected from the linear model. Thus in this cell significant directionality would only be expected at low intensities. For 12 DS neurons subjected to bicuculline, the mean control DSI was 0.35 ± 0.05, which requires I/E of 0.52. For these neurons, I/E varied between 0.30 and 0.64 and the mean was 0.45 ± 0.14, which is only marginally <0.52. For six nondirectional neurons (mean DSI = 0.17), the mean I/E is 0.25 ± 0.09 (Table 1). Thus the directional cells exhibit stronger inhibition than nondirectional cells.

 
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TABLE 1. Parameters of excitation and inhibition in directional and nondirectional tangential cells

The latency of the difference function (inhibition) minus the latency for excitation is a measure of the inhibitory delay. At the lowest and the highest intensities the delay is close to zero (as shown in Fig. 11D); whereas, at intermediate intensities it varies between 38 and 45 ms.

As noted previously the ratio of the spatial sampling interval, p, to the inhibitory delay, d, determines Vmax, the velocity for maximum directionality. In the previous study (Glantz 1994), receptive field analyses and responses to apparent motion stimuli resulted in p values of ~4° and d of 50-100 ms. For the cell described in Fig. 11, Vmax, determined with sinewave gratings, was 120°/s. Taking d (from Fig. 11D) as 40 ms and p as 4°, yields p/d of 100°/s.

The inhibitory delay was measured in 18 neurons (using the procedures shown in Fig. 10) in cells also tested for DS. In 14 of these cells, the delay varied from 28 to 148 ms as shown in the distribution of Fig. 12A. Three cells exhibited no delay and one directional cell had a delay of 288 ms. The cells exhibiting no inhibitory delay also failed to exhibit significant DS (DSI from 0 to 0.22). This result is expected for all models of DS which require a temporal asymmetry in the receptive field of DS neurons (Egelhaaf et al 1989; Glantz 1994; Reid et al 1991). Twelve of the 18 cells described in Fig. 12A exhibited DS and these neurons exhibited inhibitory delays of 28-288 ms. Excluding the outlier at 288 ms, the inhibitory delay of the directional cells was more than twice that of the nondirectional cells (Table 1).


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FIG. 12. Distribution of inhibitory delays among 18 neurons. Three cells exhibiting no delay are graphed to left of zero in histogram. Mean delay excluding zeros and outlier at 268 ms is 69 ± 32 ms for n = 13 cells.

The velocities for maximum directionality were measured in nine directionally selective neurons. Vmax was strongly correlated (r = 0.80) to the inverse of the inhibitory delay, as shown in Fig. 12B. Figure 12B () is the least squares regression function. From Vmax = p/d, the regression slope has the dimension of p and it is equal to 3.49°. Previous measurements (Glantz 1994), with sequences of flashes yielded p of 3-5°. In addition to the delay, the inhibitory functions exhibited two additional relevant features. The risetime of inhibition is about 2.5 times longer than the risetime for excitation (as in Table 1). This difference is evident in Figs. 1 and 10B. The longer risetime implies a longer time constant for inhibition and a smaller inhibitory response (relative to excitation) at higher stimulus temporal frequencies. Furthermore, as shown in Figs. 1 and 10, the decay phase of the light response is similar in the controls and during bicuculline perfusion. A similar result obtained in virtually all cells tested. This finding implies that inhibition is not prolonged relative to excitation. Thus the temporal asymmetry required for directionality must come from the early rather than the later stages of inhibition.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Several lines of evidence imply a role for a bicuculline-sensitive GABA receptor in the visual response of tangential cells. Immunocytochemical studies (Pfeiffer-Linn and Glantz 1989) indicate that GABAergic terminals of lamina monopolar neurons and GABAergic processes of medullary neurons form dense projections in the area of tangential cell dendrites. GABA depolarizes tangential cells, antagonizes the hyperpolarizing light response and antagonizes the hyperpolarizing action of acetylcholine. Furthermore, bicuculline blocks the depolarizing action of GABA and diminishes the delayed repolarization phase of the Tan1 light response. Both the effects of GABA and the antagonism of the GABA response by bicuculline are seen with synaptic actions reduced by ~90% in CoCl2. In addition to its action on tangential cells, there is evidence that bicuculline is an effective GABA antagonist at a number inhibitory synapses in crayfish nervous system (Miyata et al. 1997; Newland et al. 1996).

The results in this study indicate that GABA also plays an important role in Tan1 DS. Bicuculline reduces DS by 90% and this is largely because of an increase in the null direction response. This result is similar to findings in several systems including rabbit (Caldwell et al. 1978; Wyatt and Daw 1976) and turtle (Ariel and Adolph 1985) retinal ganglion cells, cat visual cortical neurons (Sillito 1975) and motion detectors of fly lobula plate (Schmid and Bülthoff 1988). Although the results indicate the necessity for GABA action in DS, it is possible that the relevant GABA action occurs elsewhere in the visual pathway (Nelson et al. 1994). This caveat must be weighed against the fact that tangential cells have GABA receptors (Pfeiffer-Linn and Glantz 1989) and that there is a strong relationship between the measured parameters of inhibition and DSI (Fig. 12, Table 1).

In contrast to its effect on DS, bicuculline had no effect on orientation selectivity. Thus tangential neurons differ from rabbit ganglion cells where picrotoxin (or strychnine) abolishes all of the "complex" features of the receptive field (Caldwell et al. 1978) including DS, size, and speed selectivity. GABA antagonists also eliminate selectivity in small-field motion detectors of fly lobula plate but other evidence indicated separate mechanisms for DS and size selectivity (Warzecha et al. 1993). Furthermore, DS and orientation selectivity in cat cortex also rely upon separate mechanisms (Nelson 1991). In the cortex, the dissociation may reflect the influence of intracortical excitation (Nelson et al. 1994) and the receptive field alignment of the geniculate projection neurons (Chapman et al. 1991). In crayfish, there is evidence for orientation selectivity in the lamina monopolar cells (Glantz and Bartels 1994), the first order interneurons of the visual pathway. Thus tangential cell orientation selectivity may not entail an important role for inhibition.

Bicuculline's effects on the light pulse response provides estimates of the time course and magnitude of GABA-mediated inhibition. These results are at least roughly in accord with the expectations based upon a linear model of DS. The measured inhibitory delay varies inversely with Vmax measured with drifting gratings. Furthermore, the average delay (68 ± 35 ms) is compatible with the most frequently encountered range of Vmax (30-60°/s) (Glantz 1994). The intensity-dependence of the relative inhibitory magnitude may indicate an intensity-dependence in DS (as found in flies, Pick and Buchner 1979) but this has not been systematically examined in tangential cells.

An essential feature of most models of directional selectivity is the temporal asymmetry between the excitatory and inhibitory subfields of DS neurons. The temporal asymmetry is required for the directional bias and provides a compensation for the stimulus translation time between receptive subfields. Barlow and Levick (1965) recognized that the temporal asymmetry could be achieved by either an inhibitory delay or a prologation of inhibition relative to excitation. As noted in results the onset of inhibition is delayed relative to excitation but it is not prolonged. Excitation and inhibition appear to decay in parallel after the end of a light pulse. The appearance of a delay at the onset of inhibition but none at the termination implies a nonlinearity in the inhibitory mechanism.

One aspect of the time course of inhibition, the relatively long risetime, was not anticipated by the linear model. This model assumed that the time course of inhibition should closely resemble that for excitation but for the delay and attenuation. The longer risetime implies that the inhibitory pathway contains a low-pass filter with a longer time constant than that in the excitatory pathway. This idea has been incorporated into several models of DS (e.g., Egelhaaf et al. 1989, Reid et al. 1991). As noted previously, a longer time constant for inhibition relative to excitation implies a decline in I/E and thus a decline in DS as the stimulus temporal frequency increases. Furthermore, the temporal frequency optima in the preferred and null directions of motion will be different as previously observed (Glantz 1994, Fig. 11B). Interestingly, a similar prediction was made on theoretical grounds (DeAngelis et al. 1993), with respect to DS in cat cortical neurons.

In a previous study (Glantz 1994), the temporal features of inhibition were characterized in cells exhibiting DS with paired pulses and apparent motion stimuli. The results indicated that maximum inhibition obtained at interstimulus intervals of 50-100 ms and I/E varied between cells from 0.40-1.00. In the present study, 11/12 directional neurons exhibited inhibitory delays of 28-148 ms and I/E was between 0.30 and 0.64 for all 12 cells. An important difference in the two experiments is that the apparent motion stimulus was a 3° wide bar, whereas the present study used a 24° spot covering most of the receptive field. As a consequence, I/E in the present study should be smaller because the excitatory response is elicited from all parts of the receptive field including those outside the inhibitory zone.

The above results indicate that GABA-mediated inhibition is necessary for DS in tangential cells. The inhibitory pathway exhibits a time constant that is 2.5-3 times that in the excitatory pathway. The magnitude and temporal properties of inhibition are not inconsistent with a primary DS mechanism based upon a near linear interaction of excitation and delayed inhibition. The data also lend support to the idea that the velocity-dependence of DS is intrinsic to the proposed linear mechanism. In contrast to the mechanism for DS, GABA-mediated inhibition is not required for orientation selectivity.

    ACKNOWLEDGEMENTS

  I thank B. Knight and E. Kaplan for the loan of the fiber optic lens and S. Rabinowitz for assistance in preparing this manuscript.

  This work was supported by a grant from the National Science Foundation (IBN-9507878).

    FOOTNOTES

  Received 16 July 1997; accepted in final form 3 December 1997 .

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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