Cerebral Cortex Advance Access published online on March 28, 2008
Cerebral Cortex, doi:10.1093/cercor/bhn038
Cortical Dynamics Subserving Visual Apparent Motion
Brain Research, Department of Neuroscience, Karolinska Institute, Retzius vaeg 8, S17177 Solna, Sweden
Address correspondence to Bashir Ahmed, PhD, Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Parks Road, Oxford OX1 3PT, UK. Email: bashir.ahmed{at}dpag.ox.ac.uk.
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Abstract |
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Motion can be perceived when static images are successively presented with a spatial shift. This type of motion is an illusion and is termed apparent motion (AM). Here we show, with a voltage sensitive dye applied to the visual cortex of the ferret, that presentation of a sequence of stationary, short duration, stimuli which are perceived to produce AM are, initially, mapped in areas 17 and 18 as separate stationary representations. But time locked to the offset of the 1st stimulus, a sequence of signals are elicited. First, an activation traverses cortical areas 19 and 21 in the direction of AM. Simultaneously, a motion dependent feedback signal from these areas activates neurons between areas 19/21 and areas 17/18. Finally, an activation is recorded, traveling always from the representation of the 1st to the representation of the next or succeeding stimuli. This activation elicits spikes from neurons situated between these stimulus representations in areas 17/18. This sequence forms a physiological mechanism of motion computation which could bind populations of neurons in the visual areas to interpret motion out of stationary stimuli.
Key Words: dendritic depolarization • neuron communication dynamics • visual cortex • visual motion • voltage sensitive dye
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Introduction |
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The detection of motion is an integral part of vision (Nakayama 1985
). Although there has been some progress in the physiological mechanisms the visual system uses to detect the position and motion of objects (Motter and Mountcastle 1981; Steinmetz et al. 1987; Albright and Stoner 1995), we are still far from understanding the computation of object motion. Motter et al. (1987) showed that moving objects in the parietal cortex were represented by a traveling (wave of) inhibition and excitation. Still, however it is not known how moving objects are represented in early and intermediate visual areas. Intuitively and logically, the visual system must use information related to the disappearance of a stimulus at one position and its reappearance at an adjacent position to compute motion: a result that tells the brain that an object has spatially shifted within the visual field. The topic however is more complicated, because one can perceive motion from a brief sequence of stationary images presented in different positions. This illusion is called apparent motion (AM) (Exner 1875; Wertheimer 1912). During continuous motion there is evidence that a retinal contribution to motion may exist as a time locked wave of ganglion cell spike discharge across the retina ahead of the position of a continuously moving object (Berry et al. 1999). Hence, it is difficult to discern the contributions of the brain in motion perception from the contributions of the retina. For this reason we examined AM in order to establish how early and intermediate visual areas compute motion out of a sequence of stationary objects. AM exists in primates (Newsome et al. 1986; Muckli et al. 2002; Claeys et al. 2003; Merchant et al. 2003; Zhou et al. 2003), carnivores (Pasternak 1987; van Wezel et al. 1997), and probably in all mammals.
A sequence of stationary images which induce AM can be presented over a short-range (less than a degree of the visual field) or over a longer range (several degrees). Short-range AM depends on stimulus conditions different from those causing long-range AM (Braddick 1980; Anstis and Mather 1985; Chubb and Sperling 1988). Both of these illusions are very robust in their basic form: it is impossible by conscious effort or attention to inhibit or change the direction, speed and vividness with which the stationary stimuli appear to move (Palmer 1992). As the stimuli producing AM are stationary on the retina, the brain must compute the perception of motion. It is known that neurons in early and intermediate visual areas react to long-range AM (Newsome et al. 1986; Muckli et al. 2002; Claeys et al. 2003; Merchant et al. 2003; Zhou et al. 2003). We expected long-range AM, therefore, to depend on communication within area 17 as well as long-range communication between several visual areas. As long-range corticocortical axons are excitatory (Maunsell and Van Essen 1983; Loewenstein and Somogyi 1991; Rockland and Drash 1996; Anderson and Martin 2002), one may be able to detect increases in excitation of the target neurons elicited by long-range axons (Roland et al. 2006).
We, therefore, examined the physiological basis of AM by staining 4 visual cortical areas (areas 17,18, 19, and 21) of the ferret with a voltage sensitive dye and recorded the signals from these areas under stimulus conditions that lead to long-range AM. This revealed that the long-range AM conditions tested were always associated with activations in the cortex moving in the direction of the AM. In addition, we found that these activations were always preceded by a feedback from areas 21 and 19.
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Materials and Methods |
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All experimental procedures were approved by the Institutional Animal Care and Use Committee of Karolinska Institute and were performed according to Swedish and European Community guidelines for the care and use of animals in scientific experiments and the policies of the use of animals in neuroscience research of the Society for Neuroscience, 1995.
Ten fully anaesthetized adult female ferrets (isoflurane 1%, N2O:O2, 50:50) were paralyzed (pancuronium bromide 0.6 mg/kg/h, i.v.) and artificially ventilated, expiratory CO2 and body temperature were maintained between, respectively, 3.3–4% and 37°C. The pupil was dilated (1% atropine sulfate eye drops) and a contact lens was placed over one eye, the other eye was occluded. After a right-sided craniotomy and dural resection, the animal was equipped with a pressure chamber (Optical Imaging, Rehevot, Israel) and the exposed visual cortex was stained for 2 h with the voltage sensitive dye RH 795 (Molecular Probes, Leyden, The Netherlands) at 0.5 mg/ml. Figure 1A shows the cortical region used for the optical recording.
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Visual Stimuli
Visual stimuli were presented on a computer display (Cambridge Research Systems, Cambridge, UK), refreshed at 120 Hz, placed at a distance of 57 cm from the animal. The right eye was occluded and all stimuli were delivered to the left eye. The stimulus was a white square (2° by 2°, duration 83 ms, and 120 cd/m2) presented on a uniform gray background (30 cd/m2). The square was always presented in the following positions along the vertical meridian (checked by electrophysiology) 3.5° below the horizontal meridian (lower square), at the crossing of the vertical and horizontal meridian (center square), and 3.5° above the horizontal meridian. For the stationary control conditions, the square was presented in 1 of the 3 positions at the onset time matching the AM condition (experiment 1). Figure 1B–E illustrates the 4 different stimulus conditions for AM. In experiment 1 the square is displayed successively at the 3 positions: lower, central, and top. In experiment 2, the exposure of the central and lower square and the top and central square overlaps in time (Fig. 1C). In experiment 3, the distance between the successive square positions is increased to 7°, such that 1st the lower is displayed, then the top square (Fig. 1D). In experiment 4, 1st a central square is presented, then the lower and top square is presented simultaneously. This induces a perception of the single square being split into 2 (Fig. 1D). Each stimulus trial had a 200 ms prestimulus period. A gray screen of the same average luminance as in the stimulus and control conditions was used as a baseline for the creation of difference images. The AM conditions are shown in Figure 1.
Voltage Sensitive Dye Measurements
A Wu-Tech H469-IVR camera with an array of hexagonally arranged 464 photodiode detectors and a RedShirtImagingR macroscope (RedShirtImaging, Fairfield, CT) with 2x objective were used for optical imaging. The frame rate was 1 frame every 0.61 ms. The photodiode array measured from a hexagonal cortical area of diagonal length 4.2 mm (Fig. 1A). The excitation filter was a 530 nm narrow band filter (Schott 530 VG6, Scott AG, Mainz, Deutschland) and the acquisition a long pass 610-nm filter (Schott RG 610, Scott AG). The stimulus presentation was synchronized with the electrocardiogram (ECG) signal, and respiration was stopped during stimulus presentation. During recordings the animal was placed on a vibration free table (Minus K Technology, Inglewood CA). The voltage sensitive dye RH 795 stains all layers of the cerebral cortex. Due to attenuation of the photons the signal reaching the detectors will stem mainly from the upper, supragranular layers (Kleinfeld and Delaney 1996; de Curtis et al. 1999). The pulse artifact of RH 795 is much larger than that of the new blue dyes (e.g., RH 1691) especially near pial vessels, but the signal to noise ratios of the 2 dyes are comparable in cortex away from the vessels (Civillico and Contreras 2005). The laminar staining of RH 795 is strongest for layer I of the cortex and diminishes exponentially with depth (Kleinfeld and Delaney 1996; de Curtis et al. 1999), whereas RH 1691 stains layer II the strongest (Petersen et al. 2003a; Lippert et al. 2007). Thus RH 795 might have an advantage for detecting activations in layer I, such as those that could be caused by feedback axons terminating here (Rockland and Drash 1996). Each detector channel monitored a small circular cortical area of 150 µm in diameter.
Electrophysiology and Anatomy
The action potentials of single/multiple neurons were recorded with thin tungsten electrodes (impedance range: 0.8–1.1 M
; FHC, Boudain, ME) mainly from the upper (supragranular) layers of the visual cortex. A total of 59 units responded statistically significantly to one or more of the stimuli in the AM condition. The electrode positions were marked and occasional coagulation marks were left to calibrate the depth measurements from the microdrive. A Poisson distribution was fitted to the spike trains in the prestimulus period and spikes from the background trial. Spike trains passing both the criterion of having significantly increased discharge rate compared with the prestimulus period of P < 0.01 and increased rate compared with the background condition of P < 0.01, were considered statistically significant periods of firing.
After recordings, the brain was sectioned, stained (Nissl and cytochrome oxidase) and cytoarchitectonic areal borders were marked and electrode marks identified (Innocenti et al. 2002). The sections were reconstructed and fitted to the pictures of the operative field and voltage sensitive dye recording sites, to match the electrode penetrations. The reconstruction provided a mapping of the cytoarchitectural borders between the 4 visual areas 17, 18, 19, and 21. As the cytoarchitectural border between areas 17 and 18 marks the position of the vertical meridian, we could by this independent information evaluate whether the initial electrode penetrations, for the localization of the crossing between the vertical and horizontal meridian (Fig. 1), indeed were localized along the vertical meridian. Similarly, we evaluated whether the retinotopic sites of the square stimulus (see below) were overlapping the cytoarchitectural border between areas 17 and 18.
Data Processing
The voltage sensitive dye signal from the background condition was subtracted from that of the stimulus condition trial by trial. This subtraction was done in order to remove pulse artifacts. Although the stimulus presentation was synchronized with the ECG signal, this does only guarantee that the 1st ECG spike is aligned for the stimulus and background trial, as the later ECG spikes may diverge more and more. In order for the pulse artifact subtraction to work during the whole trial, the voltage sensitive dye signal for the background condition was modified as follows. If the ECG spike for the stimulus condition arrived prior to the ECG spike in the background condition then the frames at certain time points were removed from the background condition such as to compress the background condition in time, and hence align the ECG spikes for the 2 conditions. The exact time points at which to remove the single frames were decided with linear interpolation. If the ECG spike for the stimulus condition arrived after the ECG spike in the background condition then frames were inserted in the background file in order to expand the background condition in time. If an image had to be inserted at time point tn then the actual frame inserted was the frame at time point tn–1, that is, the frame at tn–1 is copied such as to be on position tn and the following frames are shifted forward by 0.616 ms. This procedure efficiently removed the pulse artifact as seen in the data and autocorrelation in Supplementary Figure 1.
V(t)xy is the difference in fluorescence to the stimulus minus the fluorescence to the baseline gray screen, divided by the fluorescence obtained in darkness F0,xy . For 1 detector channel x,y:
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V(t)xy were averaged by adding the files and dividing by the number of files (averaging of the temporal course of the V(t)xy). In the text this average is referred to as simply V(t). Using the amplitude fluctuations in the prestimulus interval to define the noise level for each channel, the V(t)xy was thresholded at P < 0.01 of being noise (P < 0.01 1-sided, as only V(t) increases occurred in response to the stimulus conditions). In this we assumed the amplitude fluctuations to be not significantly different from a Gaussian distribution. A threshold of estimated P < 0.01 was set for each photodiode detector channel and divided by the number of channels (464) to give the Bonferroni corrected value of P < 0.01 (Fig. 2) which is used for determining the statistical significance.
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The relative amplitude was calculated for the poststimulus interval, that is, from 0 to 400 ms after the start of the stimulus as the (
V(t)xy – V(t)xymin) divided by the overall maximal amplitude – minimal amplitude (V(t)xymax – V(t)xymin), that is,
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V(t)xymin is the minimum value of V(t)xy in the poststimulus interval up to 400 ms. In the text, the index xy is suppressed and V(t)xy,relative is referred to simply as V(t)rel.
In vitro the dye signal, V(t), is a linear function of the membrane potential (Davila et al. 1973; Salzberg et al. 1973; Cohen et al. 1974; Grinvald and Hildesheim 2004). However, as the absolute dye signal depends on the amount of staining one divides the raw signal by F0, xy. Further, the dye signal must be calibrated by intracellular recordings/patch clamping. This is not possible in vivo where large populations of neurons and glia cells are stained. Furthermore, in vivo, the photons from deeper layers of cortex are attenuated and those from the upper layers are scattered. In addition, the in vivo signals, V(t)xy,stim and V(t)xy,ctl have a pulse artifact. The pulse artifact can in practice be removed (see above). Still given this, the signal may also be subjected to equipment noise and fluctuations in the number of photons due to variations in the illumination source.
Still if one assumes the noise sources are invariant and the pulsation artifact removed, it is not possible to measure depolarization and hyperpolarization in vivo. In the strict sense depolarization is an increase in the absolute value of the membrane potential of a cell. This means that depolarization is defined from the resting potential, that is the membrane potential of a neuron without any synaptic input. Consequently the definition of depolarization and hyperpolarization will not work in vivo. The V(t)xy is a difference signal between the signal introduced by the background and the square + background that is made relative due to the division by the resting light intensity, F0. If the V(t) is >0 it means that the cortex from which the signal originates is relatively more depolarized during the stimulus condition, than during the condition when only the background is exposed to the animal. If the V(t) < 0 the cortex during background condition is relatively more depolarized than it is during the AM condition. The V(t)xy will consequently indicate changes in the deporalization direction and changes in the hyperpolarizing direction, provided that the pulse artifact is removed and the noise is identical in the 2 conditions. Furthermore as the component from glia cells is moderate and has a much slower time course compared with the neuronal changes in membrane potentials (Konnerth and Orkand 1986; Lev-Ram and Grinvald 1986; Konnerth et al. 1988; Bergles and Jahr 1997), fast changes of V(t) may be ascribed to the neurons. That the V(t) is a reliable measurement of the relative changes in population membrane potentials of supragranular neurons is also verified by simultaneous in vivo measurements of the V(t) and the membrane potentials of neurons in layers II and III (Petersen et al. 2003a, 2003b; Ferezou et al. 2006). Therefore, we use the term relative depolarization for V(t) increases under the above conditions.
Retinotopic sites were defined as the 4–7 coherent small cortical areas (corresponding to the cortex monitored by 4–7 photodiode detector channels) having the maximal amplitude of V(t) close to or at the cytoarchitectonic defined borders between areas 17/18 or 19/21. The criterion was that at least 1 small area overlapped the reconstructed cytoarchitectural border. The cortical size of the retinotopic sites were estimated based on the magnification factors provided by (Manger et al. 2002; see also Fig. 1).
Detection of Wave-Fronts
The results showed that V(t) increases in the shapes of wave-fronts in amplitude plots of the cortex were associated with all conditions of AM. A wave-front is just a surface of points having the same phase. The algorithm described in Figure 3 was made to detect such wave-fronts. All wave-fronts progressed from one retinotopic site to the next retinotopic site.
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The wave-fronts appeared in 3 places of the visual cortex: 1) along the cytoarchitectural border of areas 17 and 18, and between the retinotopic site of one square and the next square; 2) along the cytoarchitectural border of areas 21 and 19, and between the retinotopic site of one square and the next; and 3) between the retinotopic site of the square at the area 19/21 border and the retinotopic site of the square at the area 17/18 border. For each of these paths, approximately 600–800 µm wide, we examined whether there was a wave-front of increased
V(t) moving between these defined sites which was time locked to the offset of the square at 1 position (30–60 ms after the offset). During its passage between these sites, the wave-front passes cortical positions monitored by the photodiode detector array channels, symbolized by the plane with the small squares in Figure 3A. To distinguish wave-front propagation from an iceberg effect, that is, progression of a general increase in all directions, we calculated the V(t)rel showing the phase of V(t). The algorithm for wave-front detection has a sliding time window of 30 ms during which it, for each amplitude level of V(t)rel above mean, plots the relation between time of arrival and distance over cortex for any progression over cortex in any direction (Fig. 3B). It then calculates the regression of time of arrival versus cortical position of progress for each of the amplitude levels (Fig. 3C) and plots the –log10P values for the slope of the regression being = 0 for all tested directions (Fig. 3D). Finally, the –log10P value is calculated for all tested levels together with the direction giving the highest –log10P value (Fig. 3E). The algorithm automatically examines all directions 0–360° in steps of 10°. For each step the –log10P value is plotted for that direction. The final direction of wave-front motion is that giving the highest –log10P value. This is the best estimate of the direction of propagation of the wave-front. As the algorithm examines each amplitude level (Fig. 3C) the positions of the wave-front surface points for that level are mapped in cortical space in time steps of 0.6 ms. If the progress/time-step is approximately identical from step to step, the points will be close to the regression line. For other directions the progress/time-step will deviate more from the regression line and, hence, will give a lower –log10P value. This will apply to any wave-front irrespective of its profile.
The time derivative of a V(t) iceberg is a wave progressing in all directions outward from the edge of the iceberg, whereas the time derivative of a directed wave-front is a unidirectional moving wave. Note that wave-fronts, in contrast to traveling waves, do not need to progress with constant velocity and amplitude. Note also that a lateral spreading depolarization out from a retinotopic site per definition is also a shallow wave-front. In this case the wave-front is circular with new amplitude levels appearing from the center or edge of the retinotopic site. For the iceberg, this is not the case, as the outward wave when the time derivative is taken of the emerging iceberg is located only where the iceberg breaks the surface, that is, at the edge of the iceberg.
Calculation of the speed of the wave-front was done from the time derivative of V(t), that is, d(V(t))/dt was calculated and then the slope of the overall regression (as in Fig. 3E) was estimated with the wave-front detection algorithm. Calculating the speed of the wave-front from the V(t) or V(t)rel tends to underestimate the velocity of propagation as higher amplitude levels tended to move more slowly.
The contribution of the wave-fronts to the total signal was calculated using a similar strategy. The V(t) was divided into 10 levels. The lowest level, showing a motion from one retinotopic site of the square to the next retinotopic site, was the magnitude that separated the wave-front from the underlying depolarization (as the wave-front was superimposed on the lateral spreading depolarization, see Results). This was done for all animals and gave a mean value of 34 ± 12%. The mean velocity of the wave-fronts was calculated from the slope of the regression line of the d(V(t))/dt from the path of the wave-front.
The differences in dynamics between an AM condition signal and the control conditions was calculated by adding the V(t)'s from the 3 control conditions in which the squares were presented individually, to form 
V(t). Then the V(t) from AM and the V(t) was normalized as described under the calculation of V(t)rel. Then the difference V(t)rel, AM – V(t)rel,sum was calculated. This difference was then differentiated, that is,
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Results |
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We calculated
V(t), that is, the difference in the voltage sensitive dye signal between luminance contrast square stimuli and the condition in which only the gray screen was shown, from the upper layers of the ferret visual cortex (Salzberg et al. 1973; Cohen et al. 1974; Grinvald and Hildesheim 2004). This signal, V(t), was sampled at a rate of 0.61 ms per frame. In this report we focus on the dynamics of the voltage sensitive dye signals associated with long-range AM elicited by a small square shown in successively different positions (Fig. 1).
In experiment 1, the ferrets were shown a short display of a static square in 1 of 3 different positions, separated by 3.5° along the vertical meridian in independent trials: lower, center, and top. This served as a control for the AM condition. In the 4th trial of experiment 1, 3 squares were shown in identical positions, but in quick succession lower–center–top (Fig. 1B). This produces a clear perception of (apparent) object motion in humans (Supplementary Video 1). The last trial was a blank screen at the same average luminance, which served as the baseline condition. The signal from this baseline condition was subtracted from the signal of the stimulus conditions to produce the voltage sensitive dye signal V(t) (see Methods). The analysis reported here was restricted to the period during which the signal was statistically significant (P < 0.01 after Bonferroni correction; see Methods). First we examined how these squares were mapped in the upper layers of the visual cortex in terms of a high V(t) increase.
As the stimuli in all experimental conditions were stationary on the retina (the animals were anaesthetized and the eye muscles paralyzed), the AM must be produced by the brain. To investigate how, one must examine the dynamics of the neuron populations in the visual areas. This is done here by measurements of relative changes in the membrane potentials of populations of neurons in the supragranular layers, V(t), of the visual areas (see Methods). There are only 3 papers dealing with the spatial and temporal dynamics in visual areas in mammals after stimulation with transient stimuli (Jancke et al. 2004; Chen et al. 2006; Roland et al . 2006). From the paper of Jancke et al. (2004) one may predict that the expected dynamics of stimulating with 2 stationary stimuli, one after another, could be approximated by a linear combination of the dynamics to the single stimuli in area 17. Directly following this, our null hypothesis is that the AM dynamics could be described in areas 17 and 18 by
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in which, VL(t) is the relative change in the population membrane following stimulation with the lower square (Fig. 1); VC(t) is the relative change in the population membrane following stimulation with the center square; VT(t) is the relative change in the population membrane following stimulation with the top square. Therefore we 1st examined the dynamics by presenting only 1 square in 1 of the 3 positions: lower, center, and top.
The 83 ms display of 1 static square produced a V(t) increase, an activation, starting at 36 ± 2 ms (mean ± SD, n = 10) following the onset of the stimulus. The activation became maximal at a site where it 1st appeared, at the location of the border between areas 17 and 18. That site was the retinotopic site of the square in the visual cortex at the area 17/18 border (Fig. 4A, Methods). With the single static square at the 3 different positions, the respective retinotopic sites were mapped along the border between areas 17 and 18, that is, along the cortex where the vertical meridian of the field of view was represented. The V(t) increase also spread out spatially from the retinotopic site such that the activation covered a larger area of the visual cortex (Fig. 4A). This lateral spread of the activation outside the immediate location of the retinotopic site has been observed for visual stimuli, even to very small ones, and has been described in detail earlier (Grinvald et al. 1994; Roland et al. 2006). Inevitably, the activation in supragranular layers, therefore, spreads to cover most of areas 17 and 18 (Roland et al. 2006). In addition, there is a weaker activation at the retinotopic site for the square at the border of areas 19/21 (yellow in Fig. 4A, middle and right). Here one can see some spatial overlap between the retinotopic sites as expected from earlier multiunit recordings in these areas (Manger et al. 2002; Cantone et al. 2005).
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When the same 3 squares, each of 83 ms duration, were presented in rapid succession (Fig. 1B), the maximal
V(t) developed at the identical retinotopic sites (Fig. 4B). However, within the time intervals between the absolute maximal V(t)s, there was a strong V(t) increase, moving from the retinotopic site of the lower stimulus toward the retinotopic site of the center stimulus (Fig. 4C). Some 34 ms after the offset of the lower square, this activation progressed from the site of the lower square toward the retinotopic site of the center square after which the V(t) at the retinotopic site for the lower square decreased (Supplementary Video 2). Similarly, after the offset of the center square, a strong V(t) increase moved from the retinotopic site of the center square toward the retinotopic site of the top square (Supplementary Video 2; Fig. 4C). The shape of these activations moving from one retinotopic site to the next, may formally be described as wave-fronts (see Methods). This description is neutral with respect to the underlying causes of these V(t) increases. The wave-fronts traversed, along the border between areas 17 and 18, from the retinotopic site of the lower square (medially) to the retinotopic site of the upper square (laterally) in the time interval in which the square appeared to move (Fig. 4C; Supplementary Videos 1 and 2). For this reason we refer to this wave-front as the 17/18 wave-front. There were no such wave-fronts in response to the stationary stimuli. The timing of the motion of this 17/18 wave-front was rather sharp, it started 34.3 ± 2.3 ms after the offset of the lower or the center square (Table 1).
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In areas 19/21, the offset of the lower square/center square also evoked an activation to the next retinotopic site of the center/top square. Although the activation here was noisier, a wave-front could be detected in most animals. This wave-front moved along the border between areas 19 and 21 as shown in Figures 2D and 7D. The wave-front in area 19/21 started 34.3 ± 2.3 ms after the offset of the lower or the center square (Table 1). In the text this wave-front is referred to as the 19/21 wave-front.
The 19/21 wave-front and the 17/18 wave-front were associated in time with yet another moving activation emanating from the retinotopic site at the area 19/21 border and moving toward the area 17/18 retinotopic site of the square (Fig. 5 at 116–132 ms and again at 206–213 ms). This V(t) increase appeared from the retinotopic site of area 19/21 as a feedback signal toward the area 17/18 border where it activated the path to the next retinotopic site along the area 17/18 border (Figs 4D and 5, Supplementary Movie 3). To characterize the phase relations of these activations over the cortex, we calculated V(t)rel (see Methods). This showed that the feedback to areas 17/18 started simultaneously with the onset of the 19/21 wave-front and prior to the 17/18 wave-front (Table 1; see also Fig. 7D). We refer to this wave-front as the motion feedback. Experiment 1, thus revealed that associated with AM, there was a 19/21 wave-front, a motion feedback to 17/18, and a 17/18 wave-front moving in the direction of AM.
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Different Forms of AM Produced Activation Wave-Fronts Moving at Different Onset Times and in Different Directions
As shown in Figure 1, our experimental protocol included a number of conditions that produced AM in humans. All conditions associated with AM were associated with statistically significant moving wave-fronts, moving from one retinotopic site to the next and with a motion feedback (wave-front detection in methods, Fig. 3 and Table 2). Figure 6A,B show further examples of the progressions of the wave-fronts calculated from the path taken by the wave-fronts from the retinotopic site of the square at its offset to the retinotopic site of the next square at its onset. It is apparent from these examples that the leading edge of the wave-fronts, that is, the d(V(t))/dt, progressed with almost constant velocity and only in this direction. Similarly, the motion feedback moved from areas 19/21 to areas 17/18, and not in the reverse direction (Fig. 6C,D; Table 2). The wave-fronts and the motion feedback could be detected in the V(t) or the d(V(t))/dt (see Methods). All 10 animals had statistically significant 17/18 wave-fronts moving in the direction of AM (P < 0.01 per animal or better, Table 2). Furthermore, in all animals, there was a statistically significant feedback from the retinotopic site in areas 19/21 toward the edge of the retinotopic site at the area 17/18 border at 29.3 ± 4.0 ms after the offset of the lower/center square (Figs 4D, 5, and 6; P < 0.01 per animal or better, Table 2). In 9 animals we confirmed that 19/21 wave-fronts progressed along the area 19/21 cytoarchitectural border (P < 0.01 per animal or better, Table 2, in 1 animal the area 19/21 cytoarchitectural border was outside the cortex monitored by the photodiode array).
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As the eye position was fixed, the squares appeared and disappeared, instantaneously, from one spatial position to the next. Accordingly, the initial on-responses of
V(t) and the retinotopic site of the squares in areas 17/18 appeared distinct. This is apparent if the time derivative of V(t), d(V(t))/dt is calculated. Figure 7A shows that the time derivatives for the 2 positions in experiment 3 are spatially and temporally separated at the area 17/18 border. That the relative depolarization at each retinotopic site at the area 17/18 border is stable in its position until the wave-fronts begin can also be demonstrated by marking the point of maximal depolarization for each time frame after the start of the stimulus (Fig. 7B). Taken over all animals and all experiments the point of maximum depolarization at the 17/18 border remained stationary within ± 150 µm until 37.1 ± 4.6 ms after the offset of the lower/center square (all experiments, mean ± SD; n = 20).
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In experiment 1, the offset of one square was timed to the onset of the next square at the new position. This did not allow one to distinguish whether it was the onset of the new square or the offset of the former square that initiated the wave-fronts. In experiment 2 our protocol allowed us to distinguish between these possibilities. The onset of the center square was timed to occur 42 ms after the onset of the lower square. Consequently, the 2 squares remained ON for 41 ms before offset of the lower square. Similarly, the onset of the top square was timed to remain ON for 42 ms before the offset of the center square (see Fig. 1C). These temporal changes in the timing of the sequence of presentations as seen in Figure 1C, had no effect on the appearance of the wave-fronts seen in Figure 7D. The wave-fronts from the retinotopic site of the lower square appeared exactly as in the 1st experiment, some 36 ms after the offset of the lower square (Table 1, Fig. 7D). Similarly, the wave-fronts traveling toward the retinotopic site of the top square, appeared on average 33 ms after the offset of the center square (Fig. 7D, Table 1). Thus the wave-fronts were time locked to the offsets and not the onsets. This is in accordance with psychophysical observations in humans (Giashi and Antis 1989).
In the 1st and 2nd experiments, the wave-fronts traversed across the cortex at the same velocity of 0.21 to 0.23 mm/ms (P > 0.5 2 tailed t-test n1 = 4, n2 = 6, Table 1). As the distance between the positions of the squares were identical in both experiments, this is perhaps not surprising. In experiment 3 we examined whether the velocity of the wave-fronts could be altered. We left out the center square and presented only the lower and upper squares with the offset of the lower square exactly timed to the onset of the top square (Fig. 1D). The result was that the wave-fronts were initiated as usual after the offset of the lower square at 33 ms and traveled in a single sweep directly to the retinotopic site of the top square (Fig. 7B). Surprisingly, the velocity of the 17/18 wave-fronts, was still 0.227 mm/ms (Table 1). There were no significant differences in wave velocities between experiments 1, 2, 3, and 4 (Table 1, analysis of variance P> 0.9).
If AM is always dependent on the visual cortex computing an activation wave-front from the retinotopic site of one stimulus to the next, a stimulation in which 1 square is followed by 2 oppositely positioned squares (experiment 4; Fig. 1E), should produce 2 wave-fronts, each traveling in opposite directions but toward the retinotopic sites of the 2 new squares. Indeed this was our finding. At 36 ms after the offset of the center square, 2 oppositely directed wave-fronts progressed in this divergent manner (Fig. 7E, Table 1). In the V(t)rel domain, 2 oppositely directed wave-fronts traveled to the retinotopic site of the lower and the top square. Although the data were noisy, a similar divergence could be seen at the area 19/21 border again preceding the 17/18 wave-fronts (Table 1; Supplementary Video 4). Thus, if a single stimulus was briefly presented and at its offset was followed by the onset of 2 stimuli but located on either side, the cortex computed 2 wave-fronts which traveled over the cortex to the retinotopic sites of the diametrically opposed stimuli. In humans this is associated with the perception of 1 object moving while being split into 2.
The initial on-responses of V(t) to the squares in areas 17/18 appeared distinct and stationary (Fig. 7A). Notably, in all conditions of AM the wave-fronts appeared 1st after the offset of the squares and moved fast between the retinotopic sites of the squares. The peak depolarization in contrast moved only slowly and after a delay vanished at the former retinotopic site, whereas the new peak increased at the next retinotopic site (Figs 4C, 5, 7B–E; Supplementary Videos 2–4).
Interarea Dynamics of AM
In experiments 1 and 4, we included control conditions in which single squares were presented in isolation at the same positions where they were presented as a sequence during the AM conditions (see Fig. 4A and Methods). When we calculated the sum of the depolarizations evoked by each of the 3 squares when presented singly, that is, the sum of the 3 files similar to those depicted in Figure 4A, the calculated amplitude of the signal V(t) for this sum in each animal was considerably larger than the V(t) for the AM after 100 ms (Fig. 8A). In order to remove the local amplitude differences, but preserve the spatial dynamics we computed the time derivative of the difference, that is, d(V(t)rel,AM – V(t)rel,sum)/dt. This difference, between the 2 conditions, showed that the AM condition was associated with a significant feedback to the area 17/18 border in the interval from 32 to 47 ms after the offset of the lower square (Figs 4D and 8B; Supplementary Video 5; Table 2).
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Relation between the Wave-Fronts and Neuronal Firing
The membrane activations in the form of the wave-fronts should be capable of firing neurons between the retinotopic sites of the squares. We, therefore, recorded electrophysiologically from 59 multiunits in areas 17 and 18. These units were significantly activated by the AM conditions (P < 0.01, see Methods). Neurons at the retinotopic sites, at the area 17/18 border (n = 22) faithfully fired an on-response in the control condition with single stimuli and in the AM conditions with no differences in onset latencies (P > 0.3, paired t-test, n = 22; Fig. 9A). For units (n = 37) located between the retinotopic sites of the lower and center squares and between the center and upper squares in areas 17/18, the spike responses were sparse or nonsignificant to the single square conditions (Fig. 9B). However, these 37 units fired significantly more in the AM condition during the time interval 110–140 ms than in any of the single square conditions (Fig. 9B; P < 0.05). For these 37 units, we compared the sum of spike trains to the AM condition with the sum of spike trains from the 3 conditions in which the squares were shown individually. This revealed that the 37 units fired significantly higher spike rates in the AM condition but only for the duration of the time interval during the passage of the wave-front (Fig. 9D; P < 0.01). Moreover, 21 of the 37 units had a mean spike rate in the AM condition which was significantly and positively correlated to the averaged V(t)rel overlying the electrode penetration. Thus, they fired in the time interval during the passage of the wave-fronts with a lag of 1–2 ms (Spearman rank correlation coefficient, r, significant at P < 0.05 or less per unit). The responses of 11 of the 37 units were negatively correlated with the wave-front V(t)rel (P < 0.05 or less per unit). Thus, the neurons at the retinotopic sites fired almost identically in the AM conditions and the single square conditions. The neurons between the retinotopic sites, mostly fired only in the AM conditions and their responses were either positively correlated or negatively correlated to the average wave-front amplitude in the time interval during its passage from one retinotopic site to the next.
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Assuming that the
V(t) originates mainly from the dendrites (Grinvald and Hildesheim 2004), the time derivative d(V(t)/dt can be regarded as proportional to the relative input driving force for the populations of neurons in the supragranular layers. For the neurons populating the space between the retinotopic sites of the square stimuli, we compared the time course of the d(V(t))/dt with the instantaneous firing rates of the neurons located here in the time interval of the wave-front passage 100–140 ms. The result was that the relative drive, that is, the mean d(V(t))/dt increase preceded the mean instantaneous firing rate increase (Fig. 10). We also compared the time of peak firing in this interval to the peak d(V(t))/dt. The result was that the peak d(V(t))/dt came statistically in advance of the peak firing (P < 0.0005; paired comparison; n = 20; the remaining neurons had no detectable peak of firing rates in this interval). These results are consistent with the idea that the wave-front passage drives the firing of the neurons in between the retinotopic sites.
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Furthermore, the neurons at the retinotopic sites of the squares at the 17/18 border fired with onset latencies not significantly different from those in the single square conditions (Fig. 9A). Thus at onset of the 2nd or 3rd square, the firing of neurons at this retinotopic site in area 17/18 did not significantly alter the spike firing of neurons at the retinotopic site of the former square.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialFundingReferences |
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In summary, the results showed that after the offset of the 1st stimulus, a moving activation wave-front appears in areas 19/21 and a motion feedback is sent to areas 17/18. Immediately, the area 17/18 wave-front starts and progresses together with the area 19/21 wave-front in the direction of AM. In the time interval during which the 17/18 wave-front traverses from one retinotopic to the next retinotopic site, the neurons located between these retinotopic sites generate spike responses. The 4 conditions that produced AM in humans, in the ferret visual cortex invariably were associated with such wave-fronts and these were triggered by the offset of the stimulus. The wave-fronts appear to correspond to the time interval eliciting AM and to the predicted speed of the AM (Supplementary Video 1; Giaschi and Anstis 1989
; Chavane et al. 2000).
A number of possibilities were examined as possible explanations for our results. First, could the wave-fronts arise from the fact that retinal ganglion cells fire slightly ahead of a continuously moving object (Berry et al. 1999) and, 2nd, could they in fact not be wave-fronts, but motion streaks. A motion streak is a "smeared contour, a tail of the cortical position of a moving object caused by temporal integration in the visual system" (Geisler 1999; Geisler et al. 2001). As our stimuli were stationary, they were unlikely to have set up a wave of excitation between successive spatial locations within the retina. Also we recorded only localized on-responses both with the voltage sensitive dye signal and electrophysiologically by recording action potentials in areas 17/18 (Figs 4–7 and 9). Finally, the squares were transiently presented with a separation of 3.5°, and the AM wave-fronts started with a 35–40 ms delay after the offset of the 1st square stimulus and not the onset of the next stimulus. A streak is defined as being behind the object moving with respect to the direction of movement. In contrast, in our case the new square had already appeared (experiment 2) when the wave-front emerged from the retinotopic site of the lower/center square and moved to the retinotopic site of the next square (Supplementary Video 3; Fig. 5).
Excluding retinal causes for the activation wave-fronts, the question is whether the physiological mechanisms of AM can be explained by a feed-forward depolarization of the supragranular layers at the area 17/18 border in accordance with our null hypothesis. From our results, the wave-fronts appeared immediately after the offset of the lower square, and not after the onset of the center square. This makes it unlikely that the On-response from the lateral geniculate nucleus via layer IV spiny stellate neurons should have produced the wave-fronts. Secondly, the sum of the signal computed from single square presentation, V(t), was consistently larger than the V(t) of the AM condition. Third, the neurons between the retinotopic sites fired significantly more during AM when compared with all single square presentation conditions (Fig. 9D). To this we have to add the statistically significant feedback (Figs 4D, 5, 7D; Supplementary Videos 3–5; Table 2). Furthermore, the direction of the increases in V(t) and V(t)rel of the motion feedback signal was toward the area 17/18 border. Moreover, this feedback signal is only present in the AM condition, and absent in the control conditions as well as the individual sum of the 3 V(t) signals (Figs 4D and 8B; and Supplementary Video 5). All these results are against the null hypothesis and hence against linear and feed-forward mechanisms. These results are also incompatible with feed-forward models of (apparent) motion (Reichardt 1961; Barlow and Levick 1965; Carandini et al. 1997).
The statistics, the accurate timing, the speed, and the averaging of the V(t) make it unlikely that the wave-fronts and the motion feedback signal could be explained as spontaneous moving depolarizations or "up-states" (Prechtl et al. 2000; Petersen et al. 2003b). These factors taken together with the fact that the ferrets were anaesthetized with their eye muscles paralyzed, make it very unlikely that the source of the wave-fronts and motion feedback signal could be the result of attention, arousal or eye movements.
The arrival of a new stimulus on the retina is associated with a feed-forward depolarization of the corresponding retinotopic site in the visual cortex and this is followed by a laterally spreading depolarization (Grinvald et al. 1994; Roland et al. 2006). The laterally spreading depolarization spreads out in all directions from the retinotopic site and attenuates after some 70 ms (Roland 2006). In experiments 1, 2, and 3, the wave-fronts associated with AM moved in the opposite direction to the initial laterally spreading depolarization caused by the onset of the next square (Figs 4–7). This was also true for experiment 4, in which the wave-fronts moved from the retinotopic position of the 1st square at offset toward the retinotopic positions of the 2 new squares, that is, again against the direction of the laterally spreading depolarizations from the new squares (Supplementary Movie 4).
Each wave-front always was unidirectional moving in the direction of AM, and also unidirectional when converted to a wave as d(V(t))/dt. This evidence suggests that it cannot be explain