Human sensory-evoked responses differ coincident with either "fusion-memory" or "flash-memory", as shown by stimulus repetition-rate effects
© Jewett et al; licensee BioMed Central Ltd. 2006
Received: 05 September 2005
Accepted: 23 February 2006
Published: 23 February 2006
A new method has been used to obtain human sensory evoked-responses whose time-domain waveforms have been undetectable by previous methods. These newly discovered evoked-responses have durations that exceed the time between the stimuli in a continuous stream, thus causing an overlap which, up to now, has prevented their detection. We have named them "A-waves", and added a prefix to show the sensory system from which the responses were obtained (visA-waves, audA-waves, somA-waves).
When A-waves were studied as a function of stimulus repetition-rate, it was found that there were systematic differences in waveshape at repetition-rates above and below the psychophysical region in which the sensation of individual stimuli fuse into a continuity. The fusion phenomena is sometimes measured by a "Critical Fusion Frequency", but for this research we can only identify a frequency-region [which we call the STZ (Sensation-Transition Zone)]. Thus, the A-waves above the STZ differed from those below the STZ, as did the sensations.
Study of the psychophysical differences in auditory and visual stimuli, as shown in this paper, suggest that different stimulus features are detected, and remembered, at stimulation rates above and below STZ.
The results motivate us to speculate that:
1) Stimulus repetition-rates above the STZ generate waveforms which underlie "fusion-memory" whereas rates below the STZ show neuronal processing in which "flash-memory" occurs.
2) These two memories differ in both duration and mechanism, though they may occur in the same cell groups.
3) The differences in neuronal processing may be related to "figure" and "ground" differentiation.
We conclude that A-waves provide a novel measure of neural processes that can be detected on the human scalp, and speculate that they may extend clinical applications of evoked response recordings. If A-waves also occur in animals, it is likely that A-waves will provide new methods for comparison of activity of neuronal populations and single cells.
The sensation transition-zone for fusion
A well established psychophysical effect is the change in sensation when repetition-rate is increased to the point where previously-sensed "individual stimuli" become "fused". In vision, flashed "stop-action" becomes a "movie" at higher flash-rates, while in audition, the sensation of "discrete sounds" comes to contain a "musical tone" when the same transient sound is repeated above about 20 S/s (Stimuli/sec). The early history of research on fusion has been summarized in these words by Kompass  :
"The first, to my knowledge, empirical contribution to this line of research was given by Lalanne (in 1876: ) who pointed out that the frequency of stimulus fusion in the tactile, auditory, and visual modality equals 18 Hz. Lalanne conjectured a common, yet unknown, mechanism behind this.
"Measuring tactile stimulus fusion, Brecher (in 1932: ) found that the critical frequency did not depend on intensity of stimulation or the cutaneous receptor density: Stimulation of the tips of tongue and fingers gave approximately the same critical frequency value as stimulation of the back or the feet. Variability between participants was very small: Individual averages of 14 participants yielded anoverall mean period of 55.3 ms (18.1 Hz) and a standard deviation of 1.2 ms between participants. This seemed surprising because it was known that other well-determinable psychological constants such as Weber fractions differ much more among participants."
A commonly-used term in psychophysiology is CFF (Critical Fusion Frequency) for the sensory transition. CFF rate has been studied as an indicator of arousal and attention and has had clinical use as a diagnostic tool for multiple sclerosis, migraine, Altzheimer's, Parkinson's and other diseases [4–11]. We will describe differences in evoked-responses as a function of stimulus repetition-rate, in which qualitatively different evoked-responses occur at repetition-rates below and above what we will call the STZ (Sensation-Transition Zone). We use the term STZ rather than CFF because referring to a rate-boundary between two phenomena in the singular implies that a single rate can be identified, and is unchanging. But a given endpoint may be affected by hysteresis, as was noted by von Bekesy [12, 13], who also found a range of auditory endpoints if intensity was held constant and frequency varied. Furthermore, in vision the CFF varies as a function of position in the visual field. For our purposes now, it is better to define the STZ as a psychophysical region where the stimulus-repetition rate may not be precisely known, may not be constant, and may depend on other stimulus parameters. We have studied stimulus repetition-rates that are on either side of the STZ. Thus, we can only describe a range of stimulus repetition-rates in which the transition occurs, not "the boundary".
A note on terminology: Since stimuli can be non-sinusoidal transients, for stimulus repetition-rate we use the units of Stimuli per second (i.e., 10 S/s). If we are referring to sinusoidal waveforms (as in the Frequency Domain), we use Hz as the units.
Technical limitations in experimentation with continuously-repeating transient stimuli
Evoked-response recordings that produce temporal waveforms have been limited to repetition-rates that provide an SI (Stimulus Interval, start -to-start) which is longer than the observed evoked-response waveform (using appropriate filtering). The consequence is that high stimulus repetition-rates have not been studied, except by means of SS (Steady-State) responses, which have important limitations (described next and in the Discussion).
SS responses are obtained using a uniform repetition-rate, which makes recovery of any time-domain transient brain-response waveform to each stimulus mathematically impossible. (For proof of this statement, see our paper on QSD .) SS evoked responses measure only the magnitude and phase of the Fourier coefficients at the stimulus repetition-rate and its integer multiples. The limitations caused by measuring only the magnitude of the Fourier coefficient (often only at the stimulus repetition-rate) may be the reason that SS evoked potentials in the auditory and visual systems [15, 16] show no change in electrical potentials that correspond to the CFF. In vision, van der Tweel et al.  looked specifically for a connection between sinusoidal SSVEPs (Steady-State Visual Evoked Potentials) and the CFF boundary measured as a function of both stimulation rate and modulation depth. They concluded that "the lack of correspondence between the results of the psychophysical studies and those obtained in electrophysiology is striking". Other studies also report a lack of correlation between evoked-potential-amplitude and subjective flicker threshold [18–20]. A study as recent as 2001 using square wave stimulation in vision also showed no particular change in the evoked potentials over the STZ . Additional information on the limitations of SS as a measure of effects of stimulus repetition-rate is in the Discussion.
QSD avoids the limitations of SS
the timing of the jittered SI pattern, and
the brain's transient response is not the uniform, identically-repeated waveform of SS. The small differences that occur in the average allows recovery of the brain's response by deconvolution of the average by the timing of the jittered SI pattern . The computational methodology for this process is called QSD (q-Sequence Deconvolution), and has been described in detail .
QSD, in brief
Because QSD will not be familiar to the reader, we provide here a brief overview of the method for those who wonder how we can now record what was previously unobservable. Details specific to the results are in the Methods Section, and further descriptions are in the original QSD paper .
The mathematics of QSD can be expressed in a few equations. The following equation states what is illustrated in Fig. 1:
that is, the recording on a channel ( (t)) is the combination of the brain's evoked-response (b(t)) convolved with the timing sequence (q(t)), and that result is algebraically summed with the noise (n(t)). Note that the noise is not convolved. Note also that all elements (b,q,n) have the same duration ((t)), which must be sufficiently long that b(t) has returned to baseline within the length of time of the q-sequence (i.e., within the SL = Sequence Length) .
In Eq1, the © symbol is used to denote the time-domain circular convolution. The recorded response (t) is a circular vector because it has been averaged on a 100% duty cycle synchronized with the cyclic, continuous, circular vector q(t). Such circular vectors can be directly converted to the frequency-domain without windowing. Thus, Eq1 becomes, in the frequency-domain:
Note that the time-domain convolution function is, in the frequency-domain, complex multiplication. We can then recover the estimated brain response in the frequency-domain [ (f)] by dividing by the frequency-domain equivalent of q(t) (which is Q(f)), as shown by the Fundamental Equation of QSD:
Animated illustrations of the differences between QSD, "SS Responses", and standard averaging will be shown in the Discussion..
"Early" results with QSD: Auditory Brainstem Responses
The relative uniformity of the waveforms at different repetition-rates in Fig. 2B is in contrast to the averaged, convolved (superposed) data shown in Fig. 2C. Note that the superposed data traces of Fig. 2C are "quasi-Steady-State" responses, i.e., they would be "Steady-State Responses" if there were no jitter. Note further that the peak-to-peak magnitudes of the convolved waveforms of Fig. 2C are not proportional to the corresponding peak-to-peak magnitudes of the deconvolved waveforms (Fig. 2B). For example, at the 120 S/s repetition-rate the convolved waveform has the highest peak-to-peak magnitude, but the deconvolved waveform at that rate is similar in magnitude to those of adjacent repetition-rates. This is one example that between-rate differences in "steady-state" responses may not reflect actual brain-response differences. (See also Discussion.)
There are several reasons to think that the waveforms of Fig. 2B are accurate. First, the direct comparison of QSD with standard averaging in Fig. 2A is good. Second, the waveforms at 80 S/s and above are all similar, despite the fact they are from different runs and that a different timing-sequence was used for each run. Third, the differences in waveshape compared with the slower rates are physiologically reasonable, showing systematic latency and amplitude changes. Fourth, there is one part of the waveform whose shape should be predictable: the pre-stimulus baseline should be relatively flat, as it is in the deconvolved waveforms (Fig. 2B). Note that there are no comparable flat portions of 6 ms duration in the convolved data of Fig. 2C (which has the same vertical scale as 2B).
Initial results with QSD: G-waves
Since the stimuli occur every 25 ms at 40 S/s, it is clear that the 80 ms-long response in Fig. 3 was overlapped in the averages before deconvolution (not shown). The upper trace of Fig. 3 shows that G0 is the "filter-integrated" ABR that is observed when G-waves are recorded with a 30–120 Hz passband. Fig. 3 shows that both brainstem and higher neural levels can be recorded in the same sweep. It will be apparent that this panoptical view also occurs with A-waves (see Results).
The letter "G" can serve as a mnemonic for "gamma" since the period of the G-waves is within the gamma-range of the EEG. We label these "G-waves", rather than the AMLR, because they were obtained at a stimulus repetition-rate which caused the responses to be overlapped. We define the term "G-wave" to be any auditory evoked-response within a latency range of 10 ms to about 100–125 ms (assuming the 30 – 120 Hz passband). It will be seen in Results that when the highpass filter has a lower value, the waves continue on for considerably longer.
Overview of results section
We present our preliminary data on A-wave human evoked-response waveforms. We show visA-waves, audA-waves, and somA-waves, and find both differences and similarities in waveform as a function of stimulus repetition-rate. We also offer evidence that these unusual waveforms are not artifacts of the QSD calculations.
We first show the effect of stimulus repetition-rate on visA-waves. There are systematic differences as a function of repetition-rate (especially above and below STZ). We next show audA-waves, including examples of the variation of these waveforms, within day, and between days. While we do not have enough data for statistical analysis, there is evidence that the differences as a function of repetition-rate are not due to "selective data selection" by the authors.
In the next section we show that visA-waveforms are similar in shape to known "after-discharge" visual responses, even though visA-waves are obtained with continuous stimulation at high repetition-rates. We then show that differences in somatosensory somA-waves are seen above and below STZ.
To assuage worries that these new phenomena are artifacts, we then show the evidence we have so far accumulated that these responses are not generated by the QSD method, and hence need to be seriously considered as a new measure of brain activity.
Using the QSD method with a filter passband where the highpass is below 30 Hz, we have found oscillatory waves some of which have periods in the "alpha" range of the EEG. The first author could not resist naming these "A-waves". A-waves are operationally defined as those waveforms obtained with a highpass less than 120 Hz, that have a duration longer than the SI used to evoke them, i.e., the stimulus repetition-rate is fast enough that the responses overlap. The definition of "A-waves" does not require that the waveform have oscillations with a period within the alpha-EEG range, although many A-waves at supraSTZ rates have had such oscillations. A-waves without sustained oscillations have been recorded in response to subSTZ stimulus repetition-rates. Note again that the term "A-waves" only implies that the responses are longer than the SI, and does not require that the response have sustained oscillatory components, though many do.
Effects of stimulus repetition-rate on visA-waves
Only the first 800 ms after the stimulus are shown.
The waveforms are normalized to an equal height by using different vertical scales on the traces.
The waveforms of 20 S/s through 90 S/s have been moved to the right, so that the second negative valley will align with the same valley in the 15 S/s waveform, as shown by the solid vertical line. The 10 S/s and 15 S/s waveforms have not been moved.
(The choice of the second negative valley was somewhat arbitrary, being chosen because the wave is large, present in all of the traces, and seemed to be the onset of consistent oscillations following it.)
Note the considerable similarity in shape of the visA-waveform across the rates at and above 15 S/s, though the amplitudes do vary (refer back to Fig. 4). The shift in latency necessary to bring about the alignment can be seen by the blank space at the start of the traces that were moved. The shift means that wave-peak latency shortens as the repetition-rate increases. Note also that the waveform at the rate of 10 S/s was different from that seen at all the other rates. We were surprised that even at a repetition-rate as slow as 10 S/s the response was longer than the SI, thus requiring QSD to obtain this response. Another surprise was finding such long-duration waveforms correlated to stimuli being delivered at such high rates. The amount of overlap can be seen in the convolved averages, which is accessed from the Fig. 4 legend.
Effect of stimulus repetition-rate on audA-waves
Variation in audA-waves, within-day, between-day
In Fig. 9 we show audA-wave run-to-run differences in 4 young-adult subjects, 3 male, and one female, at two repetition-rates (below and above STZ). In Fig. 9, note the similarities of the waveforms between rates (within a subject) up to about 50 ms, with marked divergence thereafter. G-waves are visible as the overlapped waves before 50 ms in Fig. 9. G-waves are much less affected than are the A-waves by both the repetition-rate differences and the run-to-run differences. We tentatively consider the latency interval between 50 and 80 ms to be the transition between G-waves and oscillatory A-waves in auditory responses.
We hypothesize that the variation before 50 ms is due to the low-frequency EEG "noise" within the A-wave passband. (When we have studied auditory G-waves with a passband of 30–120 Hz, this degree of variability is not present. Hence, the EEG contribution to the waveform after 50 ms is presumably about the same as the variation before 50 ms.) The A-wave oscillations start at a latency about 80 ms. A notable feature in Fig. 9 is the clear difference between the waveforms of 15 S/s and 30 S/s after about 50 ms. These differences are notably larger than the run-to-run differences. So, we consider that the repetition-rate differences are not likely to bedue to run-to-run differences, despite the absence of calculated statistical probabilities. (The data was collected in exploratory fashion without the predesigned form required for the rigorous statistical analysis that we hope to provide in later papers.)
Although in Fig. 9 we show only the first 300 ms of these audA-waves, in 2 of the 4 subjects the audA-waves continued past 500 ms (not shown). Consequently, the original (convolved) data is highly overlapped, as was shown with respect to the audA-waves of Fig. 8 (see Legend). The first positive peaks at 30 S/s in Fig. 9 are about 93 ms apart and the first negative peaks are about 110 ms apart. These two periods would correspond to about 11 Hz and 9 Hz respectively, placing them within the "alpha-frequency" range of the EEG.
The correspondence between audA-wave peaks and the peaks of traditional AEP waves is unclear at this point. As is known from the ABR, if experimental conditions are changed, the waveform at a fixed latency after the stimulus can be due to different neural generators. Clearly changing the stimulus repetition-rate is a changed experimental condition that may also change the neuronal contributions to observed peaks. So, we choose, at this time, not to use previous peak-naming conventions based solely on latency.
Somatosensory responses (somA-waves)
It is unclear whether persistent oscillations in somA-waves will be obtained in future recordings, since they do not occur in Fig. 11C. It is notable that in this 74 yr old male subject, the visA-waves (Fig. 11A) and audA-waves (Fig. 11B) also recorded do not show the prolonged oscillations, either. Thus, the absence of oscillations in somA-waves may be a function of the age of the subject, or some other factor.
Similarities between A-waveforms and "after-discharge"
The first 240 ms shows the early waves in the VEP. Some of our visA-wave recordings show them and some do not (see Figs. 4 and 6); we do not understand why. The similarity between Fig. 12A and 12B suggests that we are recording the early events in the VEP, despite the overlap (SI = 33 ms at 30 S/s).
We now turn to the oscillations with about a 110 ms period that are shown at the slower time axis in Fig. 12A, in the latency range of 240–1340 ms. The visA-waves of Figs. 4 and 6 also show such oscillations out to about 1000 ms. So, our QSD-derived visA-waves show activity to high-rate stimulation that has been previously known only with respect to slow rate stimulation.
One might imagine the persistent waveform after the end of stimulation (A1 and B1 in Fig. 13) as being due to length of the individual responses and the decline as due to the diminishing amount of overlap. One can also imagine that, at the start of a train of stimuli at a rapid repetition-rate, there would be summation of the overlapping visAwaves of Fig. 4, which might have a shape similar to the "onset response" in Fig. 13B2. However, we know that at the beginning of a train of stimuli that the responses cannot be identical. There must be a transitionfrom the subSTZ waveform to the supraSTZ waveform at the startof a prolonged stimulation at a (nearly) uniform repetition-rate. This is shown in the next Section.
The morphing of subSTZ waveform to supraSTZ waveform
A run is recorded with slowly repeated pairs of stimuli (the time between stimuli inthe pair being short, but the time from start-of-pair to start-of-next-pair is long), where the overlapped responses are shown in Fig. 14A, along with the timing of the two stimuli, 1 & 2. To remove the overlap in the response to the pair we record the next run.
A run using single stimuli at a slow rate gives the response shown in Fig. 14B.
The response to just the second stimulus (Fig. 14C) is then moved to the left (note stimulus mark in Fig. 14D), so as to permit easy comparison with other responses relative to the time of the stimulus that generates them.
A third response is obtained using high-rate stimulation, where the SI in the continuous stimulation is the same as the timing between the stimuli in the pair. The response would be deconvolved using QSD, to obtain the response to each stimulus (Fig. 14E).
Finally, the three responses are compared:
The response to a single stimulus (Fig. 14B), which must be the same as the response to the first stimulus in a stimulus train (if the repetition-rate is slow enough to be equivalent to "no prior stimuli").
The isolated response to the second stimulus (Fig. 14D), which is different because it was affected by the prior (first) stimulus.
The response to a sustained repetition of the stimulus (Fig. 14E), which is the "steady-state" response due to stimuli separated by that period (which is also the period between the pair of stimuli in Fig. 14A). This response must occur at some point in the stimulus train if the stimulus train is long enough.
The Solid trace is the response to Dau-chirp stimuli jittered at a mean rate of 15 S/s (same sequence as used for timing the start-to-start of the pairs). The jittered SI was required because the audA-waves are longer than the SI.
The Dotted trace in Fig. 15 is the response to the second stimulus of a pair ofDau-chirps where the pair had a fixed interval of 14 ms from start-to-start. The inter-pair interval (start-to-start) was jittered around a mean interval of 67 ms (= 15 S/s), so that it was the same rate and pattern as for the response to the single stimulus (Solid trace). Because of the overlap of the first response to the second, it was necessary to do the subtraction, as diagrammed in Fig. 14C and then shift as in Fig. 14D.
The Dashed trace in Fig. 15 is the deconvolved response to a Dau-chirp, recorded with a stimulus repetition-rate of 70 S/s (which has the same period [14 ms] as the separation of the pairs (Dotted trace).
Clearly, Fig. 15 shows that a pair of stimuli are insufficient to evoke the audA-waveshape obtained from sustained rapid stimulation, although some "elements" of the sustained waveform begin to develop by the second stimulation. For example, note the opposite polarities of the two responses (solid and dashed lines) at about 90 ms and at 120 ms, with the dotted trace having intermediate values. Thus, the "full" audA-wave takes some number of repetitions of the stimulus before reaching an asymptotic waveshape. This finding has implications for the kinds of neuronal mechanisms that are involved in generation of A-waves, and also justifies our not trying to overlap visA-waves to mimic increasing or decreasing "after-discharges" as shown in Fig. 13.
Evidence against artifactual waveforms
With any new technique, especially if it presents unusual results, it is reasonable to wonder whether artifacts are created of such magnitude as to produce the unexpected. We provide now a number of different lines of evidence against artifactual generation of waveforms by the QSD technique.
A second line of evidence is that very similar-appearing waveforms can be obtained from different-appearing convolved data, as shown in the convolved data used to find the waveforms of Fig. 8 (see Legend to access the convolved data file).
The time-domain average for the 3.0 s SL is shown in the Middle trace of Fig. 17. The periodicity-peaks with the shortest inter-peak time occur at the stimulus repetition-rate. No consistent 100 ms periodicity is seen in the Middle trace, in marked contrast to the deconvolved waveform in the Top trace. In the Bottom trace of Fig. 17, we shut off the auditory stimulation, and averaged for the same length of time as taken in recording the Middle trace. When that average was deconvolved, the result was the Bottom trace, which shows no evoked-response, providing further evidence to that shown in Fig. 16.
A-waves, being a new evoked-response phenomenon, raise a number of issues, none of which can be definitively settled in an introductory paper such as this. Instead, we hope to indicate in this discussion what questions the findings generate, and in what ways these new phenomena might be useful.
Trivial coincidence, or tantalizing clue?
Mindful that "coincidence implies causality but does not prove it", the consistency of waveform differences in the visual, auditory, and somatosensory systems on either side of the STZ provides powerful motivation for producing some wide-ranging speculation. We demonstrate our primary speculation by means of Fig. 20, which shows a group of grey dots of different sizes. The presentation is steady (at the refresh rate of the screen you are watching). You are now going to see the same screen flashed, where one of the dots will move back and forth a distance of about its radius.
Additional file 5: MovieC. This is a QuickTime presentation of the dots of Fig. 20, at a high rate. (MOV 2 MB)
Next try MovieC (see Fig. 20 legend). The repetition-rate for MovieC is 12 S/s with a 100% duty cycle. Hopefully you now answer the question in the affirmative, and that it is easier to see the dot move under the conditions of Button C. Note that seeing the dot move did not require any conscious effort, or any prior use of "attention". The detection of the moving dot is automatic (and presumably a relatively low-level of extraction of a changing stimulus embedded within a background that appears unchanging because of fusion).
What are the neurophysiological mechanisms that underly this psychophysical experience?
What functional role might such mechanisms play? Since we cannot immediately answer the first question, let's start with the second.
With regard to the "functional role" that this neurophysiological mechanism plays, a "scene-presentation" with most elements having a repetition rate above STZ provides a means to rapidly identify a change in the visual field. Whereas, when one is presented with the same stimuli at a subSTZ repetition-rate, it is very difficult to identify the dot that moves, even when one knows which dot to look at. For ease of speculation about neurophysiological mechanisms, let us assume that a "single" stimulation is followed by a single firing of the cells in the early part of the response (as is possible if the stimulus magnitude is adjusted to be moderate – neither near threshold nor near saturation – and the stimuli are brief). Certainly this kind of firing can be found at sensory cells in the PNS (Peripheral Nervous System). We assume, in this case, that the firing of subsequent post-synaptic cells in the CNS (Central Nervous System) is not at a slower rate than the rate at which the PNS cell is being driven.
A firing rate below the STZ,
A firing rate, though above the STZ, that is changing. (Our attempts to define "Figure" and "Ground" have always lead to either obvious or subtle circular definitions; we therefore will purposely avoid rigor.)
The effect of Fig. 20 relies on the Ground being presented above STZ, while the Figure is presented below STZ. If we imagine that the sensation of "fusion" involves the detection of unchanging "sameness", then there must be a memory of the immediate past, and an estimate for the duration of that memory can be made from our experiments. Based on our data, we would roughly estimate the longest duration of this "fusion-memory" is about 80 ms for peripheral vision, and about 60 ms for the auditory system. (Based upon the work of Lalanne  and Brecher  the value would be 56 ms [the period of 18 Hz].) That is, we predict that any sensory inputs that are repeated at shorter unchanging intervals than some short interval, will give the "supraSTZ response", where the word "unchanging" implies "less than the just-noticeable-difference for that stimulus repetition-rate" (a criterion apparently met by a low-jitter QSD because there is a fusion effect despite the jitter).
When comparing the quality of "fusion-memory" with "flash-memory", fusion-memory is much more accurate for some aspectsof the stimulus. For example, even though there is memory of a single flash of Fig. 20, it is not possible to remember enough detail to determine that one of the dots is changing position. The accuracy of fusion-memory is shown when something changes in an otherwise "stationary" scene. Look out a room-window at a scene in which nothing seems to be changing. A small movement of something in any part of the scene is rapidly noticed, even when the details of the scene are complex, unfamiliar, or even random. Somehow fusion-memory retains the "current-state" of the pattern of sensory-input, so that change is readily detected.
On the other hand, if change in a scene is sufficiently slow, it will go unnoticed – a neural phenomenon which is utilized by many predators who use slow approaches to prey, at a speed below that which triggers an "alerting" response in the sensory system of the prey.
To allow you to compare the accuracy of fusion-memory, with flash-memory, we offer a demonstration in the auditory system. For this demonstration, the sounds must be played through loud speakers, not headphones. If you have a stereo computer system, space the computer's speakers about 1.3 meters, or more, apart. Put one speaker at least 0.5 meter closer to you than the other, so that the "Dau-chirps" will appear to originate between the speakers, but closer to the near speaker (even though the "Dau-chirps" in both speakers will actually occur simultaneously). If you have difficulty observing the effects described, then try either moving the speakers a bit farther apart, placing yourself more asymmetrically relative to the speakers, and/or trying it in a smaller space, such as a closet. (The senior author has added the mention of the closet here, so that if someone finds you listening to buzzing sounds in a closet, you can produce written evidence to them that you are not totally crazy.)
To start stimulation at 2 S/s (uniform) use the following link [see Additional file 2] Adjust the intensity to be comfortably loud. Also set the audio-player to "loop" so that the sound plays continuously if it is not doing that.
Point your finger to the spatial location from which the sound seems to originate. Now rotate your head, left and right, over about a 60° range. Note that despite movements of the head relative to the speakers, the "location" of the sound is unchanged, and easily indicated by your pointing finger. Further, note that the subjective quality of the sound does not change with this head movement. Confirm the same observations by moving your head closer and farther from the speakers by about 15 cm. You have experienced what we call "flash-memory".
Now start stimulation at 100 S/s (uniform) using the following link [see Additional file 4]. Although the sound is raspy, a low-pitched tone is perceptible, in addition to higher-frequency timbre. Repeat the observations you made after pushing the 2 S/s button. Does the quality or loudness of the stimulus sensation change with even small changes in head position or rotation? (If so, return to "2 S/s (uniform).mov" to verify that you cannot hear these differences at the slower rate. [see Additional file 2]).
Is the accuracy of your locating the "source" the same as with the low rate, or has the "location" broadened? You have experienced what we call "fusion-memory".
(You might remember this the next time you encounter the sound of a solitary cricket's "chirp" and find it difficult to physically locate the cricket solely by its sound. The senior author presumes that the frequency of the cricket chirp is above your STZ, but somehow starts and stops without energizing flash-memory in predators (while having a different effects in other crickets). Another example is the lack of "location effect" for a sub-woofer in a multi-speaker sound system, where the sounds are cyclic repetitions that are supraSTZ.)
Did you notice that when you were listening to "100 S/s (uniform)" (fusion-memory) that you could hear the "glitch" when the sound-player on the computer reaches the end of the track and takes a moment to loop to the re-start? If not, try again: [see Additional file 4]. This is the very feature of the sensory input that fusion-memory is very good at detecting. Can you hear the glitch listening to "2 S/s (uniform)" (flash-memory)? The same timing "glitch" is there, too, but not detectable by flash-memory. You can verify this: [see Additional file 2].
These effects are important in that the subjective differences observed can be hypothesized to be due to differences in memory functionality between the shorter fusion-memory (at repetition-rates above STZ) and the longer flash-memory (at repetition-rates well below STZ). We hypothesize that these psychophysical differences are due to differences in neural processing which are reflected in A-wave differences. Another important aspect of these differences is that up to now time-domain waveforms from evoked-response research have been limited to those observable at subSTZ stimulus repetition-rates – so the conclusions from such research only apply to flash-memory. The issue of SS studies of supraSTZ rates is discussed later, in a separate section.
To demonstrate that the psychophysical effects are still present even though there is a small amount of jitter in the stimulus-intervals, as is required by QSD, we offer the same stimuli here, but with stimulus repetition-rates which are jittered 12% as compared with the uniform rates. "2 S/s (jitter)." [see Additional file 1] "100 S/s (jitter).mov" [see Additional file 3]
Flicker-fusion and visA-waves
Also in Fig. 22, we provide some sequences with increased jitter, not used in our experiments, to show the psychophysical effects of increased jitter. Note that the sounds of the non-jittered (uniform) 40 S/s [see Additional file 48] tend to form a low-frequency tone. That tone is less in the 12% jitter that we used [see Additional file 58]. At higher jitters the tone is gone – 24% [see Additional file 59] and 36% [see Additional file 60]. It is also missing with the MLS sequence [see Additional file 57]. This observation suggests to us that we were lucky to have not started with a larger jitter, and that future A-wave research needs to verify whether the waveforms differ at percentage jitters less than 12%.
We did not try to find an A-waveform "at the fusion-point" in our studies because such an endpoint might be highly variable, could differ with intensity, and, like threshold measurements, could involve many long runs. Our choices of stimulus repetition-rates were based upon guesses in the hopes of staying on either side of the STZ. It may be that the unusual audA-waveform at 15 S/s in Fig. 8 is within the STZ since it is unlike the waveforms at rates either above or below it. There may well be interesting changes occurring within the range of about 12–20 S/s, as indicated by Fig. 23. In 1936, v. Bekesy reversed the usual procedure and kept the stimulus magnitude constant while varying frequency while searching for a fusion threshold, using a closed ear-canal stimulator . He had this to say about fusion threshold:
"Careful examination revealed that the auditory threshold for low tones reflects the quantal character of neural processes. Thus if the frequency of the alternating pressures was changed slowly, and without any variation of magnitude [emphasis added], from 2 to about 50 cps, it was possible to observe that the loudness and the pitch did not vary continuously, but were altered in a stepwise manner.
"This discontinuity was most clearly perceptible in the region of 18 cps. As the higher frequencies were approached, there appeared a sudden increase in loudness, corresponding approximately to a doubling of the sound pressure. At the same time there was a doubling of pitch; the number of pulses, which were separately perceptible below 18 cps, suddenly became doubled, and the whole sensation became fused and acquired a tonal character (Brecher) . This tone was still extremely rough, and the roughness gradually declined as the frequency was raised further. This frequency therefore can properly be designated as the threshold of fusion (Brecher) . It is practically the same for all the sensory modalities..."
We might expect that A-waves might be affected by these factors, although the A-waves we have shown are all recorded "above threshold" in terms of intensity and repetition-rate.
The relevance of QSD to psychophysical research on the phenomenon of "fusion" is that QSD provides a means of correlating observable brain activity with psychophysical endpoints (if the endpoints can be adequately defined and determined). Whether the correlation will be exact remains to be seen. But it is clear that in those research or clinical areas where flicker-fusion shows interesting and/or useful results, QSD may make a contribution. For example, visA-waves might be helpful in those patients in whom the subjective CFF measure is unreliable, such as Parkinson's [4–6]. Even where the patient's CFF is reliable, the objective measure of visA-waves by QSD might augment or replace the psychophysical measurement of CFF in a variety of clinical conditions, such as migraine, Alzheimer's, reading disabilities, hypertension, drug side-effects, and visual deficits [7–11].
It is of interest that there seems to be a latency "shift" in the peaks of the larger A-waves when comparing waveforms at rates above and below the STZ (Figs. 4, 6, 8, 9, 10, 11). Such a shift can only be known for certain by research which shows which peaks in the subSTZ and supraSTZ waveforms are functionally comparable. But, for the purpose of this section, let's assume that such latency differences are present. Given that we have associated subSTZ and supraSTZ waveforms with different memories (fusion-memory and flash-memory), it is but a small additional leap to consider whether the differences which are a function of repetition-rate are somehow connected with some mechanism that we will imagineas being similar to the spike-timing dependent plasticity of LTP (Long-Term Potentiation) and LTD (Long-Term Depression).
At excitatory cortical synapses, induction of synaptic plasticity is dependent both on the rate and the timing of input activities. While experimental protocols for study of these phenomena tend to emphasize the timing of activation rather than the rate, it is clear that both are jointly responsible for the induction of synaptic plasticity [25, 26]. While this plasticity is generally studied and conceptualized with respect to changes lasting minutes to hours, in our model we assume that the mechanisms that trigger these longer effects may also trigger shorter memory mechanisms, as well. So, we note that, with respect to rate, LTP occurs with higher stimulation rates (e.g., 60–100 S/s), while LTD occurs with low rate stimulation (e.g., 13–20 S/s). The sign of plasticity (LTP or LTD) is dependent on the temporal order of synaptic activity relative to the back-propagation of the action potential. This temporal order might be affected by the latency-shifts we are assuming. The magnitude of the shift that we "eyeball" between the subSTZ and supraSTZ waveforms is 70–80 ms. This is of an appropriate size to move from the LTD window (75–50 ms before the action potential) to the LTP window (10–15 ms after the action potential) [25, 26]. Hence, it is conceivable that cellular mechanisms could be triggered by the latency shift that distinguishes subSTZ and supraSTZ responses, and by implication might distinguish fusion and flash memories.
The effects triggered by the LTP/LTD mechanism with respect to fusionmemories would be predicted to be very short (if not enhanced by attention, emotions, etc.), such as less than 75 ms.
We wonder whether the time needed from the start of a rapid stimulus train, to develop the supraSTZ waveform (Fig. 15) should have some equivalent time at the cellular level. Such equivalent time might be the time necessary for activity in the dendritic tree, at higher input frequencies, to induce a prolonged depolarization in the cell that, together with continued synaptic activity, induces LTP .
Since the senior author hypothesizes that evoked-responses obtained at stimulus repetition-rates above about 6 S/s are almost entirely due to action potentials, he cannot resist commenting here that an increased peak latency of A-waves at supraSTZ firing rates (as controlled by stimulus repetition-rate) could be a measure of timings of the action potentials causing the back-propagation required for the "LTP-triggered" model suggested here. If so, then some aspects of the timing of these cellular processes could be detected and measured on the human scalp for research and clinical purposes. Note, however, the large number of "if's" needed to reach this notion.
The senior author also conjectures that the EEG may be the "ground" brain activity which registers the "current status" of unchanging sensory inputs via A-wave oscillations time-locked to the steady firing-rate of an given sensory input (as a function of the steady stimulus intensity at each sensory input, independently). When a changing sensory input results in a markedly-uneven firing rate, then the "figure" thus identified is rapidly analyzed with the brief A-wave responses (below STZ). The analysis involves associative memory.
Does the large amount of "ground" activity affect the affect the associative memory search?
Is "ground" the "context" of the resulting association?
The "oscillatory nature" of A-waves
A-waves indicate a new source of data about brain activity, obtained by a technique that can directly stimulate and record sustained oscillations of more than 1000 ms after each stimulus in a rapid stimulus train. This data may contribute to global theories of brain function that utilize "oscillations" as a generalization underlying many aspects of brain functioning, as described in several books [27–31]. Some articles report research on brain oscillatory behavior based upon EEG or ERP data, e.g., [32–34] while other articles describe theoretical approaches, e.g. [35–37]. Our data suggest that QSD methods may be applied across a considerable range of studies directed toward understanding neural oscillations, with the hope that this new approach may complement and deepen the interpretation of previous results and hopefully uncover new phenomena.
A-waves as probability functions
At first thought, an "oscillation" might seem to indicate repetitive firing from neurons driven by the stimuli. Indeed, we described such neurons with reference to Fig. 21-Right. There is much evidence to indicate that PNS cells can be driven in timing with the repetitive stimuli, as can CNS cells that are innervated by such cells. But as one ascends the neurons of a sensory system, towards the cortex, it becomes more and more difficult to achieve a simple one-to-one correspondence between the timing of a simple stimulus and the timing of the cellular response. Such observations are relevant to considerations of what cellular activity underlies scalp-recorded A-waves.
If such a model is accepted, what is the significance of a negative potential, in contrast to a positive potential, as a measure of probability? The senior author has previously shown that an AP (Action Potential) will produce one polarity at far-field electrodes when the AP is initiated, and the opposite polarity at the termination of the axon [38–40]. Since these potentials are dipoles rather than quadrupoles, these events are more easily detected at distant electrodes than is conduction along the axon (which can be quadrupolar). The consequence would be that the half-cycle time of the A-waves would be the conduction time from the initial segment to the axonal termination, as measured in a population of neurons. If the AP from neuron "A" activates neuron "B" and neuron B's AP travels subsequently in the opposite direction to the AP from neuron A, the initiation of the AP in B will have the same polarity as the termination of the AP of B. In such a case, the repetitive oscillations seen in A-waves are consistent with cyclic activity between two brain areas, such as could arise from thalamo-cortical or cortical-cortial reciprocal connectivity.
"Can the brain really do THAT?"
We have received this type of comment from reviewers, and we feel it important to describe the limitations that affect any waveshapes that are obtained by averaging. It should be clear that an average may not represent any particular individual datum. Consider that although the mean number of children per family may be 2.3, there is no family with that number of children. This fact does not negate the usefulness of the mean value, but does limit its interpretation to the population of families rather than to any one family. So, while it is easy to imagine that the mean evoked-response occurs with every stimulus, this may not be the case. As mentioned in the previous paragraph, in the case of an initially poor signal-to-noise ratio, it is not possible to detect signal variation from run-to-run variation (see Appendix of QSD paper ). So the interpretation of the "meaning" of a waveform in terms of the neuronal generators which created it during a run of repeated stimuli may be different for different evoked responses. Note that these statements refer to averaging, which is the first step in QSD. Deconvolution of the average is the next step, but does not change the basic problem that has already been generated by the average. Said in another way: QSD shares with averaging of evoked-responses the same ambiguities with regard to whether the average-waveform occurs with each stimulus or not.
"How can a nonlinear brain response be detected by a purely linear mathematical scheme?"
"Steady-State" Potentials compared with QSD waveforms
Starting in the early 90's, phenomena and theoretical excitement about the functional role of cortical oscillations (alluded to in the previous section), there was an expansion of the range of application of the SSVEP (Steady-State Visual Evoked Potential). The SSVEP was combined with cortical localization and topographic analysis, where the SSVEP was used as a "probe stimulus" that revealed activity in various areas of the brain under conditions of sensory and cognitive processing [41–44]. In the probe-SSVEP studies, the SS (Steady State) response amplitude is considered to vary inversely with intensity of processing in any area, according to the "processing capacity model" put forward by Papanicolaou . The "spare" resources available to process the probe response go down as task processing load increases. This may have the same physiological mechanism as the well studied inverse variation of alpha amplitude with increased processing activity found in "Evoked Response Desynchronization" studies [46–52]. Several established researchers have developed the SS technique with their own technical variations and created new experimental designs [48, 53, 54] to apply the SSVEP to diverse fields of study [43, 50–52, 55, 56], with clinical applications to areas such as migraine [57, 58], schizophrenia [55, 59], and Attention Deficit Hyperactivity Disorder [60, 61].
Oscillatory neural processing over all parts of the cortex,
Cognitive processing from early sensory discrimination, recognition, and attentional processing, to complex cognitive tasks,
Working and long term memory as related to decision processes, and
Motor output sequencing and coordination. At the root of all this capability and these techniques is the use of the SS stimulation.
The stimulus intervals in QSD are jittered, whereas in the SS response they are uniform.
The stimuli used in this paper, are brief, whereas "probe-SSVEP" stimulation uses sinusoidal stimulation.
Although these differences make direct comparisons between published results and ours problematic, the overlapped waveform average (i.e., the "raw data" before deconvolution) approximates to the SS average which would be obtained using our brief stimuli (with a uniform repetition-rate). For this reason, we call it the qSS (quasi-Steady-State) average. The peak in the frequency-domain at the stimulus repetition-rate in the qSS average has a peak that is equivalent to the SSVEP magnitude. So, if experimental conditions are similar, the results of the two methods can be reasonably compared in the frequency-domain.
We will now visualize the SSVEP results that would be obtained recording this brain response. As we have already proven , averaging overlapping waveforms is temporal convolution. In the frequency-domain, temporal convolution becomes just complex multiplication of the magnitude of the Fourier coefficients at each frequency in the frequency-spectra of the two circular vectors. So, if we want to know the frequency-domain result if the temporal waveform of Fig. 4(30 S/s) were uniformly convolved, we need only multiply the frequency-spectrum of the signal(Fig. 26) by the frequency-spectrum of the uniform stimulus repetition-rate, which is a comb filter. The "comb filter" is so-named because the identical-height amplitudes in the frequency-spectrum of the uniform stimulus pattern look like the teeth of a gap-toothed comb. Comb filters for five uniform repetition-rate stimulation sequences, are shown at the bottom of Fig. 26.
Because the frequencies between the "teeth" of the comb filter are zero, the magnitude of the product resulting from the multiplication of zero times the visA-wave amplitude, no matter what it is, will be zero. Hence, there are no "results" from these frequencies, only from those frequencies that have "teeth". So we need only look at the products that will result at these frequencies. Starting with the comb for a uniform stimulus repetition-rate of 5 S/s, the first product will be very small, the second very large, the third about 50% of the second, the fourth but a quarter of the third, and the rest being as small or smaller than the first. These products are the totality of the frequency-domain information available from the time-domain average from the uniform repetition-rate. This limited information is too sparse to recover the time-domain waveform from the frequency-domain data.
At 10 S/s, only two frequencies (10 and 20 Hz) contribute significantly to the products.
At 15 S/s and stimulus repetition-rates above 15 S/s, only the product at the stimulation frequency has much magnitude.
At a repetition-rate of 30 S/s, no frequencies of the response from 8–29 Hz (that were actually occurring when the brain was stimulated at 30 S/s) would contribute to the result!
If the usual practice in SS analysis were done, namely that only the product at the frequency of stimulation is used, then the data obtained from the 6 runs at the bottom of Fig. 26would show marked variation in amplitude even though the actual brain response is the same in every run!
The response changed, or
The response didn't change (i.e., a different part of the response is being probed).
In consequence, the inherent information limits in SS data as a function of repetition-rate must be recognized. This error occurs when the experimental variable is repetition-rate. If the repetition-rate is held constant while some other variable is changed, then changes in the magnitude of the product at the stimulus repetition-rate may indicate changes in brain activity if the change in the experimental variable causes no changes in the general waveshape (time-domain), but only changes the magnitude of the entire brain response. But the waveshape must be determined using QSD, in order to validate such SS data.
(Technical note: The critique centered on Fig. 26 has not included the 1/N factor in Fourier Transformations, nor whether the magnitude of the comb filter varies with repetition-rate because of repeated use of the same sweep length in the average. The general conclusion would be the same, should these have been included.)
Because the limitations imposed by data collection at a uniform rate are important when considering SS data, we have animated the differences between SS analysis and QSD, as shown in demonstrations accessed from the Legend of Fig. 27. In each of these demonstrations, in the lower left is shown a red waveform which is the brain's response to the stimulus (time-domain). On the lower right (in the box) in red are the magnitudes of the brain's response in the frequency-domain. The vertical lines indicate the frequencies of the comb filter. Across the top is the time-domain data that will occur from repeated stimulation, as computed from the convolution of the comb filter and the brain's frequency-domain magnitudes. Recall that in an SS recording, this waveform cannot be deconvolved. On the other hand, in a QSD recording this waveform approximates to the SS recording, so we call it "qSS" (quasi-Steady State), and it can be deconvolved, as shown in the middle left. This waveform (middle left) is the time-domain waveform that occurs either as deconvolved brain response in QSD, or as a 500 ms window for SS.
As you watch the SS animations, you can see that as the repetition-rate changes, different frequencies of the brain response (lower right) make up the convolved waveform (across the top). As the stimulus repetition-rate gets faster and faster, the frequencies "probed" by the comb-filter become less and less, and the waveform of "the response" (middle left) becomes more and more simple, until it is just a sine wave (when only a single tooth of the comb-filter is within the frequency of the brain response).
In contrast, as you watch the QSD animations, you will see that the frequencies that are "probed" are always numerous because of the jittered sequence of the stimuli. Note that the deconvolved waveform recovered by QSD (middle left) is the same as the brain's response. In the absence of noise the two waveforms would be identical. Since you might not believe that we were actually computing the deconvolved waveform, we added some noise within the passband, so that the waveform changes slightly.
On repeated viewings, the reader can verify that whereas in the SS animations the convolved waveform becomes simpler and simpler as the repetition-rate increases, in the QSD animations there is continued complexity in the convolved waveform (across top). It is this complexity that the QSD method utilizes to recover the brain's response. The use of a uniform repetition-rate destroys such information.
What we did not find
Like the dog that did not bark in one of the Sherlock Holmes' mysteries, what we did not observe may also be of some importance. Although oscillations with periods in the alpha-band were often observed, no prolonged or sustained oscillations in the gamma band were seen. Note that in Fig. 3, G-waves show only 1.5 "cycles" in the gamma frequency range (between the peaks G0 and G2, and between the valleys G1.3 and G3). The G-waves are not prolonged oscillations, as seen in the A-waves. If the data is recorded with a passband of 30–120 Hz (as we have done for G-waves) then there can be summation of the 25 ms periods of the G-waves (peaks adding to peaks) since the larger A-waves are filtered out. If so, the decreased amplitude above and below 40 S/s with this passband can easily be due to peaks adding to valleys. Since we have recorded with an "open passband" in Fig. 19, one can see that the "G-wave portion" of the audA-wave recording is very small. So, if the "alpha-rate oscillations" seen in the open passband are removed by a high-pass filter, then the remaining waves may sum in the time-domain, as just described. If the observations of this explanation are replicated, then the lack of gamma activity in our recordings will be viewed in retrospect as not surprising. In which case we would have to conclude that 40 Hz may not be a critical stimulus repetition-rate to whatever part of the CNS that is responding in synchrony to our jittered stimuli . Note however, that this critique applies only to "40 Hz evoked responses" recorded from the scalp, not to data from single cells or cell groups. Thus, we hypothesize that it is possible for scalp-recorded evoked-responses to seem to support single unit data, when the "support" is actually artifactual, based upon a fortuitous period between peaks in the ABR-AMLR, not upon cortical firings. Note further, that these comments do not apply to any induced oscillations which the stimulation may have caused and which we did not measure. ( What is notable is that some of our supraSTZ stimulus repetition-rates are in the gamma range. Thus, our results can be interpreted as showing long, synchronized "alpha waves" due to prolonged stimulation at gamma rates . However, the waveforms obtained at these rates are not unique to "gamma-rate"stimulation since similar waveforms were recorded to "below-gamma" rates. Our only sure conclusion is that QSD methodology offers a new way to study stimulus repetition-rate effects in sensory systems.
The data presented here is exploratory in nature, but the results, if confirmed in further research, could have important implications for both clinical electrophysiology and neuroscience.
For clinical electrophysiology, finding new CNS functionality that can be measured by scalp potentials opens new paths for detection of clinical abnormalities, even before the basis of the potentials is fully understood.
that stimulus repetition-rate can distinguish two different "modes" of CNS processing;
that these modes may differentiate ground from figure;
that these modes require two different memory mechanisms: fusion-memory and flash-memory;
that the character of these evoked-responses indicates a need for animal experiments in which both single-unit studies and evoked-response recordings are simultaneously recorded while switching "modes";
that these results show details that cannot be found with SS methods; and
that these findings provide a bridge between psychophysics and electrophysiology, in which the same phenomenon can be studied in the same subjects, at the same time.
Further information about this paper and topic is available online at .
Adult subjects were recruited and gave informed consent in accordance with a protocol approved by our Institutional Review Board. One 17 yr old adolescent was also recorded after his parents gave their informed consent. None of the subjects had a history of epilepsy in themselves or family members. We often studied subjects who were being recorded under various other projects. Subjects normally came to the laboratory for more than one visit. Each visit could last for up to 5 hours. Short breaks and meals were scheduled in the session, and subjects were encouraged to request a break if fatigued. All data was coded with a two-letter identification that was unrelated to the subject's name, and these codings were used in this paper.
The subject's hearing was verified to be normal with a pure-tone audiometer, and vision by means of a Snellen chart. For visual studies we recorded from 6 subjects. We tried a large variety of stimulations in an exploratory mode, and took more than 100 runs, each requiring at least 10 min. From this set, the visA-waveforms in this paper were from 2 females and 1 male, age range 17–52 yrs. For auditory studies the data shown was selected from about 300 data runs, recorded in 21 subjects ranging in age from 21–73 years. The auditory runs usually took 40 min each. From this set, the audA-waveforms in this paper were from 5 males and 1 female, ages 17–26 yrs. For somatosensory recordings, one subject, age 74 years, was studied using electrical median nerve stimulation.
The subjects sat semi-reclined in a chair with the head supported, to relax the neck muscles. The stimulus intensity was always comfortable, and subjects were asked to inform us if the stimuli seemed too bright or too loud.
Standard tin scalp electrodes were placed at C3'-O2 (where C3' is located halfway between C3 and Cz). Electrode paste was used for good contact after cleaning the skin with mildly abrasive gel on a Q-tip applicator. Potentials were amplified using battery-powered amplifiers from SA Instruments (Gain = 50,000) and then fed to the A-D converter (Swissonics) which connected to the computer via light-pipes. Recordings were acquired on a Mac G4 computer running MAX/MSP software, with A-D sampling at 48 kSamples/sec per channel, 24 bit accuracy with 100% duty cycle. The A-D was clock-coupled to the D-A (stimulus) (also 24 bit, at 48 kSamples/sec), and the D-A output also had 100% duty cycle. The data were stored direct-to-disk, for offline analysis. The usual recording time for visual stimulation was 10 min, during which 375 "sweeps" of a 1.6 sec timing sequence were placed on the computer disk. At a 10/sec stimulus repetition-rate, this is 6,000 stimuli, and at 90/sec it is 54,000 stimuli. The numbers would be 4 times larger for a 40 min auditory run. The usual filter settings of the amplifier were 1–500 Hz.
For flash stimuli we used a Shimpo battery-powered digital stroboscope (model DT-315A) with an external trigger. The data-acquisition computer triggered the strobe with q-sequences. The strobe was mounted outside of one wall of the Faraday chamber, with the flash directed at a square aperture in the chamber. The subject listened to music via stereo headphones with source-electronics outside of the chamber, while fixating on a 1 cm diameter colored push-pin to the right or left of the square aperture, the aperture being 157 cm from the subject's eyes. When fixating on the pin the center of the white paper was 24° from the fovea. The aperture was covered with a blank piece of white paper to diminish the intensity. The dimensions of the white paper was 12 × 12 cm, which was 4.4° at the viewing distance (2.2° from center to edge). The mean luminance of the square was 1 cd/m2, with a range of 0.1 cd/m2 on repeated measurements.
Auditory stimuli were delivered by an Etymotic ER-2 tubephone, that used comfortable soft-sponge rubber ear-canal inserts. The intensity of the stimulation was adjusted according to the subject's hearing threshold and comfort level. Usually the stimuli were at an intensity of about 65 dB SL (threshold determined at slow rates). Stimuli were wither monaural 100 s clicks or increasing-frequency "Dau-chirps" , which covered a range of 500 Hz to 15 kHz and lasted about 6 ms. Zero time was set at the end of the chirp (when all the VIIIth nerve fibers are predicted to be in synchrony)but did not include the 1 ms delay in the ER2 tubing. Dau, et al  have shown that these chirps synchronize the VIIIth nerve firings better than other stimuli such as clicks or tone-pips.
Somatosensory stimulation was by electrical pulses 0.1 ms long, from a Grass S4 stimulator with stimulus isolation unit, at an intensity sufficient to cause the thenar muscles to contract. The stimulation was not painful. ( Stimulus sequences were previously determined as described in the QSD methods paper . The sequences are given in Table 1 [see Additional file 16]. [The overall SL was chosen so as to cancel 60 Hz line interference when outputted at 48 kHz  In most of the cases the Q-magnitudes for a sequence were all above unity in the passband. In 5 cases the Q-magnitudes were below unity for one or two frequencies – in which case in the deconvolution the Q-magnitudes were "adjusted" to unity . The sequences that were adjusted in this way were (see [see Additional file 16]): 12, 16, 35, 40 S/s (Fig. 8), and 20 S/s (Figs. 4 &6). This adjustment made no significant difference in the appearance of the time-domain waveform.
Data was analyzed offline, first by averaging the raw data from the disk, and then by deconvolution calculations, as described in the Background, and in the QSD-methods paper . Data analysis used a Mac G5 with our own software, which was incorporated into an IGOR (Wavemetrics) environment. Filtering was done after deconvolution, convolving the circular filter with the circular data. The filter was a Blackman-Harris window with the 3dB points placed at the stated passband limits. Thus, the q-sequence had Q-magnitudes greater than unity for the range of the passband, and usually for a few additional frequencies in each transition-band (at which the filter attenuation was the least). This filter minimized ringing in the time-domain.
There may be problems in reading some .mov files in this article when using the web browser safari. Such problems can be overcome by downloading to disk, or by using another browser.
List of Abbreviations and Definitions
- © :
the symbol used in this paper to denote the time-domain circular convolution.
- ABR :
Auditory Brainstem Response
- AEP :
Auditory Evoked Potential
- AMLR :
Auditory Middle Latency Response
- audA-wave :
- A-wave :
an evoked-response waveform with a latency starting at about 80–100 ms and whose duration is longer than the SI of the sustained stimulus repetition-rate used to obtain it. That is the data is overlapped by the high stimulus rate.
- B(f) :
b(t) transformed to the frequency-domain.
- (f) :
the estimated brain response which contains noise, in the frequency-domain.
- b(t) :
the brain's evoked-response in the time-domain.
- (t) :
the estimated brain response, which contains noise, in the time-domain.
- CFF :
Critical Fusion Frequency. The repetition-rate at which individual sensations"fuse" into a steady sensation. See also STZ.
- CNS :
Central Nervous System
- flash-memory :
memory that occurs when the SI is greater than about 150 ms placing it as a subSTZ repetition-rate.
- fusion :
the psychophysical property when individual stimuli in a sequence cannot be distinguished. NOTE that for a given stimulus there can be a range of fusion- boundary frequencies because different aspects of the stimulus may fuse at different frequencies.
- fusion-memory :
memory that occurs when the SI is shorter than about 80 ms, placing it as a supraSTZ repetition-rate.
- G-waves :
evoked-responses with a latency of about 10–100 ms after the stimulus when obtained with a repetition-rate that overlaps the responses.
- Hz :
Hertz. Cycles per second. In this paper it is used only in relationship to sine waves. (see S/s)
- jitter :
variation in SI in a q-sequence.
- LTP :
- LTD :
- N(f) :
n(t) transformed to the frequency-domain.
- n(t) :
noise that contributes to the recorded signal in the time-domain.
- PNS :
Peripheral Nervous System
- Q(f) = q(t) :
transformed to the frequency-domain.
- q-sequence :
a Quasi-periodic timing sequence which has a small percentage jitter and meets special frequency-domain constraints.
- QSD :
- qSS :
quasi-steady-state. The potentials obtained by averaging when the stimulus repetition-rate is varied by only a small percentage by a q-sequence.
- q(t) :
the time-domain binary representation of the q-sequence as a series of one's and zero's.
- SI :
Stimulus Interval (start-to-start) between successive stimuli. The SI is the time interval between two successive stimuli in a q-sequence. [To be distinguished from ISI (not used in this paper) which is the InterStimulus Interval (end-to-start.]
- S/s :
Stimuli per second. A measure of stimulus repetition-rate. In this paper this unit is used, not Hz (q.v.).
- SS :
"Steady-State". This implies a uniform stimulus repetition-rate with zero jitter, as contrasted with qSS.
- SSVEP :
"Steady-State Visual Evoked Potential"
- STZ :
Sensation-Transition Zone. The range of stimulus repetition-rates in which the sensation of "individual stimuli" changes to a "continuity".
- subSTZ :
sub Sensation-Transition Zone, i.e., a stimulus repetition-rate that is belowthe Sensation-Transition Zone.
- supraSTZ :
supra Sensation-Transition Zone, i.e., a stimulus repetition-rate that is above the Sensation-Transition Zone.
- VEP :
Visual Evoked Potential
- visA-wave :
- (f)= v(t):
transformed to the frequency-domain.
- (t) :
the recorded activity from the scalp, including both brain activity and noise, in the time-domain.
We gratefully acknowledge the funding support of NIH via grants RR014002 from the National Center for Research Resources, MH054922 from the National Institute of Mental Health, and NS36880 from the National Institute of Neurological Disorders and Stroke.
The QSD methodology and equipment was developed under NIH grants MH054922, NS26209, DC00489.
Gilbert Goodwill was the moving force behind introducing calculations in the frequency domain into QSD and using simulated annealing for q-sequence searches.
Dr. Helmut Riedel of the University of Oldenburg kindly provided the software for analysis of q-sequences.
We thank BMC Neuroscience for publishing the paper, and especially for creating a publishing format that allowed us to demonstrate important phenomena by means of attached audio and video files. The ability to introduce a large number of figures and files (97 for this paper) is a substantial increase in the quantity of communication of scientific results. We consider that the quality has also increased, but it will be the readers who will be the ultimate judges of this.
- Kompass R: Universal temporal structures in human information processing: a neural principle and psychophysical evidence. Psychophysics Beyond Sensation: Laws and Invariants of Human Cognition. Edited by: Kaernback C, Schroger E, Muller H. 2004, Mahwah, NJ: Lawrence Erlbaum Associates, 451-480.
- Lalanne L: Sur la duree de la sensation tactile [On the duration of tactile sensations]. Note Comptes Rendus de l'Acadenue des Sciences Paris. 1876, 39 (2–3): 1314-1316.
- Brecher GA: The emergence and biological significance of the subjective time unit perceptual moment. (in German: Die Entstehung und biologische Bedeutung der subjektiven Zeiteinheit – des Moments. Zeitschrift fur vergleichende Physiologie. 1932, 18: 204-243.
- Maskin MB, Riklan M, Chabot D: Effects of "short-term" versus "long-term" L-Dopa therapy in parkinsonism on critical flicker frequency. Percept Mot Skills. 1974, 38 (2): 455-458.View ArticlePubMed
- Riklan M, Levita E, Misiak H: Critical flicker frequency and integrative functions in parkinsonism. J Psychol. 1970, 75: 45-51.View ArticlePubMed
- Riklan M, Misiak H, Levita E: Cryothalamectomy in parkinsonism. Effects on critical flicker frequency. J Nerv Ment Dis. 1972, 154 (1): 19-21.View ArticlePubMed
- Kowacs PA, Piovesan EJ, Werneck LC, Fameli H, Zani AC, da Silva HP: Critical flicker frequency in migraine. A controlled study in patients without prophylactic therapy. Cephalalgia. 2005, 25 (5): 339-343. 10.1111/j.1468-2982.2004.00861.x.View ArticlePubMed
- Vianya-Estopa M, Douthwaite WA, Pesudovs K, Noble BA, Elliott DB: Development of a critical flicker/fusion frequency test for potential vision testing in media opacities. Optom Vis Sci. 2004, 81 (12): 905-910.PubMed
- Gutherie AH, Hammond BR: Critical flicker fusion frequency: relation to resting systolic blood pressure. Optom Vis Sci. 2004, 81 (5): 373-376. 10.1097/01.opx.0000135084.16018.ac.View ArticlePubMed
- Wildberger H, Junghardt A, Neetens A, van den Ende P: [Critical (foveal) flicker fusion frequency (CFF) is helpful in differential diagnosis of organic lesions from orthoptic amblyopias]. Klin Monatsbl Augenheilkd. 1998, 212 (5): 311-313.View ArticlePubMed
- Schmitt JA, Riedel WJ, Vuurman EF, Kruizinga M, Ramaekers JG: Modulation of the critical flicker fusion effects of serotonin reuptake inhibitors by concomitant pupillary changes. Psychopharmacology (Berl). 2002, 160 (4): 381-386. 10.1007/s00213-001-0993-y.View Article
- von Bekesy G: Low-frequency thresholds for hearing and feeling. Experiments in Hearing. 1960, New York: McGraw Hill, 257-266.
- von Bekesy G: Low-frequency thresholds for hearing and feeling (in German: Uber die Horschwelle und Fuhlgrenze langsamer sinusformiger Luftdruckschwankungen). Annalen der Physik. 1936, 26 (3): 554-556.View Article
- Jewett DL, Caplovitz G, Baird B, Trumpis M, Olson MP, Larson-Prior LJ: The use of QSD (q-sequence deconvolution) to recover superposed, transient evoked-responses. Clin Neurophysiol. 2004, 115 (12): 2754-2775. 10.1016/j.clinph.2004.06.014.PubMed CentralView ArticlePubMed
- Rager G, Singer W: The response of cat visual cortex to flicker stimuli of variable frequency. Eur J Neurosci. 1998, 10 (5): 1856-1877. 10.1046/j.1460-9568.1998.00197.x.View ArticlePubMed
- Herrmann CS: Human EEG responses to 1–100 Hz flicker: resonance phenomena in visual cortex and their potential correlation to cognitive phenomena. Exp Brain Res. 2001, 137: 346-353. 10.1007/s002210100682.View ArticlePubMed
- van der Tweel LH, Lunel HF: Human visual responses to sinusoidally modulated light. Electroenceph Clin Neurophysiology. 1965, 18: 587-598. 10.1016/0013-4694(65)90076-3.View Article
- van der Tweel LH: Relation between psychophysics and electrophysiology of flicker. Docum Ophthalmol. 1964, 64: 287-304. 10.1007/BF00160581.View Article
- Kamp A, Smyth CN: Frequency analysis of the EEG. Medical Electronics. 1960, 128-131.
- Kamp A, Vliegenthart W: Sequential frequency analysis: amethod to quantify event related EEG changes. Electroenceph Clin Neurophysiol. 1977, 42: 843-846. 10.1016/0013-4694(77)90240-1.View ArticlePubMed
- Herrmann CS, Senkowski D, Rottger S: Phase-locking and amplitude modulations of EEG alpha: Two measures reflect different cognitive processes in a working memory task. Exp Psychol. 2004, 51 (4): 311-318. 10.1027/1618-322.214.171.1241.View ArticlePubMed
- Burkard RF, Hecox KE: The effect of broadband noise on the human brainstem auditory evoked response. I. Rate and intensity effects. J Acoust Soc Am. 1983, 74: 1204-1213. 10.1121/1.390024.View ArticlePubMed
- Hall JW: Handbook of Auditory Evoked Responses. 1992, Boston: Allyn Bacon
- Geissler HG: The temporal architecture of central information processing: Evidence for a tentative time-quantum model. Psychol Res. 1987, 49: 99-106. 10.1007/BF00308674.View Article
- Abbott LF, Nelson SB: Synaptic plasticity: taming the beast. Nature Neuroscience supplement. 2000, 3: 1178-1183. 10.1038/81453.View Article
- Nelson SB, Sjostron PJ, Turrigiano GG: Rate and timing in cortical synaptic plasticity. Phil Trans R Soc Lond B. 2002, 357: 1851-1857. 10.1098/rstb.2002.1162.View Article
- Traub RD, Jefferys JGR, Whittington MA: Fast Oscillations in Cortical Circuits. 1999, Cambridge,: MIT Press
- Haken H: Brain Dynamics. 2002, Berlin: Springer
- Destexhe A, Sejnowski TJ: Thalmocortical Assemblies. 2001, Oxford: Oxford Univ. Press
- Basar B: Brain Function and Oscillations. I: Brain Oscillations. Principles and Approaches. 1998, Berlin: SpringerView Article
- Basar B: Brain Function and Oscillations. II: Integrative Brain Function. Neurophysiology and Cognitive Processes. 1999, Berlin: SpringerView Article
- Schack B, Rappelsberger P, Vath N, Weiss S, Moller E, Griessbach G, Witte H: EEG frequency and phase coupling during human information processing. Methods Inf Med. 2001, 40: 106-111.PubMed
- Schurmann M, Basar E: Alpha oscillations shed new light on telation between EEG and single neurons. Neurosci Res. 1999, 33: 79-80. 10.1016/S0168-0102(98)00116-3.View ArticlePubMed
- Basar E, Basar-Eroglu C, Karakas S, Schurmann M: Brain oscillations in perception and memory. Int J Psychophysiol. 2000, 35: 95-124. 10.1016/S0167-8760(99)00047-1.View ArticlePubMed
- Meyer C, van Vreeswijk C: Temporal correlations in stochastic networks of spiking neurons. Neural Comput. 2002, 14: 369-404. 10.1162/08997660252741167.View ArticlePubMed
- Volk D: Population oscillations in a discrete model of neural netowrks of the brain. BioSystems. 2001, 63: 35-41. 10.1016/S0303-2647(01)00145-9.View ArticlePubMed
- Mongillo G, Amit DJ: Oscillations and irregular emission in networks of linear spiking neurons. J Comput Neurosci. 2001, 11: 249-261. 10.1023/A:1013775115140.View ArticlePubMed
- Deupree D, Jewett DL: Far-field potentials due to action potentials traversing curved nerves, reaching cut nerve ends, and cross boundaries between cylindrical volumes. Electroenceph clin Neurophysiol. 1988, 70: 355-362. 10.1016/0013-4694(88)90054-5.View ArticlePubMed
- Jewett DL, Deupree D: Far-field potentials recorded from action potentials and from a tripole in a hemicylindrical volume. Electroenceph clin Neurophysiol. 1989, 72: 439-449. 10.1016/0013-4694(89)90049-7.View ArticlePubMed
- Jewett DL, Deupree D, Bommannan D: Far-field potentials generated by action potentials of isolated frog sciatic nerves in a spherical volume. Electroenceph clin Neurophysiol. 1990, 75: 105-117. 10.1016/0013-4694(90)90158-G.View ArticlePubMed
- Silberstein RB, Schier MA, Pipingas A, Ciorciari J, Wood SR, Simpson DG: Steady-state visually evoked potential topography associated with a visual vigilance task. Brain Topogr. 1990, 3 (2): 337-347. 10.1007/BF01135443.View ArticlePubMed
- Silberstein RB, Ciorciari J, Pipingas A: Steady-state visually evoked potential topography during the Wisconsin card sorting test. Electroencephalogr Clin Neurophysiol. 1995, 96 (1): 24-35. 10.1016/0013-4694(94)00189-R.View ArticlePubMed
- Patterson J, Owen CM, Silberstein RB, Simpson DG, Pipingas A, Nield G: Steady state visual evoked potential (SSVEP) changes in response to olfactory stimulation. Ann N Y Acad Sci. 1998, 855: 625-627. 10.1111/j.1749-6632.1998.tb10633.x.View ArticlePubMed
- Silberstein RB, Nunez PL, Pipingas A, Harris P, Danieli F: Steady state visually evoked potential (SSVEP) topography in a graded working memory task. Int J Psychophysiol. 2001, 42 (2): 219-232. 10.1016/S0167-8760(01)00167-2.View ArticlePubMed
- Papanicolaou AC, Johnstone J: Probe evoked potentials: theory, method and applications. Int J Neurosci. 1984, 24 (2): 107-131.View ArticlePubMed
- Pfurtscheller G: Functional brain imaging based on ERD/ERS. Vision Res. 2001, 41 (10–11): 1257-1260. 10.1016/S0042-6989(00)00235-2.View ArticlePubMed
- Srinivasan R, Nunez PL, Silberstein RB: Spatial filtering and neocortical dynamics: estimates of EEG coherence. IEEE Trans Biomed Eng. 1998, 45 (7): 814-826. 10.1109/10.686789.View ArticlePubMed
- Nunez PL, Silberstein RB: On the relationship of synaptic activity to macroscopic measurements: does co-registration of EEG with fMRI make sense?. Brain Topogr. 2000, 13 (2): 79-96. 10.1023/A:1026683200895.View ArticlePubMed
- Nunez PL, Westdorp AF: The surface Laplacian, high resolution EEG and controversies. Brain Topogr. 1994, 6 (3): 221-226. 10.1007/BF01187712.View ArticlePubMed
- Muller MM, Picton TW, Valdes-Sosa P, Riera J, Teder-Salejarvi WA, Hillyard SA: Effects of spatial selective attention on the steady-state visual evoked potential in the 20–28 Hz range. Brain Res Cogn Brain Res. 1998, 6 (4): 249-261. 10.1016/S0926-6410(97)00036-0.View ArticlePubMed
- Morgan ST, Hansen JC, Hillyard SA: Selective attention to stimulus location modulates the steady-state visual evoked potential. Proc Natl Acad Sci U S A. 1996, 93 (10): 4770-4774. 10.1073/pnas.93.10.4770.PubMed CentralView ArticlePubMed
- Muller MM, Teder-Salejarvi W, Hillyard SA: The time course of cortical facilitation during cued shifts of spatial attention. Nat Neurosci. 1998, 1 (7): 631-634. 10.1038/2865.View ArticlePubMed
- Burkitt GR, Silberstein RB, Cadusch PJ, Wood AW: Steady-state visual evoked potentials and travelling waves. Clin Neurophysiol. 2000, 111 (2): 246-258. 10.1016/S1388-2457(99)00194-7.View ArticlePubMed
- Nunez PL, Wingeier BM, Silberstein RB: Spatial-temporal structures of human alpha rhythms: theory, microcurrent sources, multiscale measurements, and global binding of local networks. Hum Brain Mapp. 2001, 13 (3): 125-164. 10.1002/hbm.1030.View ArticlePubMed
- Silberstein RB, Line P, Pipingas A, Copolov D, Harris P: Steady-state visually evoked potential topography during the continuous performance task in normal controls and schizophrenia. Clin Neurophysiol. 2000, 111 (5): 850-857. 10.1016/S1388-2457(99)00324-7.View ArticlePubMed
- Muller MM, Teder W, Hillyard SA: Magnetoencephalographic recording of steady-state visual evoked cortical activity. Brain Topogr. 1997, 9 (3): 163-168. 10.1007/BF01190385.View ArticlePubMed
- de Tommaso M, Stramaglia S, Marinazzo D, Guido M, Lamberti P, Livrea P: Visually evoked phase synchronisation changes of alpha rhythm in migraine. Correlations with clinical features. Neurol Sci. 2004, 25 (Suppl 3): S283-284. 10.1007/s10072-004-0311-7.View ArticlePubMed
- de Tommaso M, Sciruicchio V, Bellotti R, Castellano M, Tota P, Guido M, Sasanelli G, Puca F: Discrimination between migraine patients and normal subjects based on steady state visual evoked potentials: discriminant analysis and artificial neural network classifiers. Funct Neurol. 1997, 12 (6): 333-338.PubMed
- Line P, Silberstein RB, Wright JJ, Copolov DL: Steady state visually evoked potential correlates of auditory hallucinations in schizophrenia. Neuroimage. 1998, 8 (4): 370-376. 10.1006/nimg.1998.0378. %L Silberstein314.View ArticlePubMed
- Silberstein RB, Farrow M, Levy F, Pipingas A, Hay DA, Jarman FC: Functional brain electrical activity mapping in boys with attention- deficit/hyperactivity disorder. Arch Gen Psychiatry. 1998, 55 (12): 1105-1112. 10.1001/archpsyc.55.12.1105.View ArticlePubMed
- Carter JD, Farrow M, Silberstein RB, Stough C, Tucker A, Pipingas A: Assessing inhibitory control: a revised approach to the stop signal task. J Atten Disord. 2003, 6 (4): 153-161.View ArticlePubMed
- Sammer G, Blecker C, Gebhardt H, Kirsch P, Stark R, Vaitl D: Acquisition of typical EEG waveforms during fMRI: SSVEP, LRP, and frontal theta. Neuroimage. 2005, 24 (4): 1012-1024. 10.1016/j.neuroimage.2004.10.026.View ArticlePubMed
- Perlstein WM, Cole MA, Larson M, Kelly K, Seignourel P, Keil A: Steady-state visual evoked potentials reveal frontally-mediated working memory activity in humans. Neurosci Lett. 2003, 342 (3): 191-195. 10.1016/S0304-3940(03)00226-X.View ArticlePubMed
- Keil A, Gruber T, Muller MM, Moratti S, Stolarova M, Bradley MM, Lang PJ: Early modulation of visual perception by emotional arousal: evidence from steady-state visual evoked brain potentials. Cogn Affect Behav Neurosci. 2003, 3 (3): 195-206.View ArticlePubMed
- Silberstein RB, Danieli F, Nunez PL: Fronto-parietal evoked potential synchronization is increased during mental rotation. Neuroreport. 2003, 14 (1): 67-71. 10.1097/00001756-200301200-00013.View ArticlePubMed
- Gray M, Kemp AH, Silberstein RB, Nathan PJ: Cortical neurophysiology of anticipatory anxiety: an investigation utilizing steady state probe topography (SSPT). Neuroimage. 2003, 20 (2): 975-986. 10.1016/S1053-8119(03)00401-4.View ArticlePubMed
- Muller MM, Hillyard S: Concurrent recording of steady-state and transient event-related potentials as indices of visual-spatial selective attention. Clin Neurophysiol. 2000, 111 (9): 1544-1552. 10.1016/S1388-2457(00)00371-0.View ArticlePubMed
- Dau T, Wegner O, Mellert V, Kollmeier B: Auditory brainstem responses with optimized chirp signals compensating basilar-membrane dispersion. J Acoust Soc Am. 2000, 107: 1530-1540. 10.1121/1.428438.View ArticlePubMed
- Larson-Prior LJ, Hart MT, Jewett DL: Neural processing of high-rate auditory stimulation under conditions of various maskers. Neurocomputing. 2004, 58–60: 993-998. 10.1016/j.neucom.2004.01.157.View Article
- Regan D: Human Brain Electrophysiology. 1989, Amsterdam: Elsevier
- Ciganek L: The EEG response (evoked potential) to light stimulus in man. Electroencephalogr Clin Neurophysiol. 1961, 13: 165-172. 10.1016/0013-4694(61)90132-8.View ArticlePubMed
- Ratliff F: Inhibitory interaction and the detection and enhancement of contours. Sensory Communication. Edited by: Rosenblith W, Barlow HB. 1961, New York: Wiley, 183-203.
- Abratech Corporation Website. [http://www.abratech.com]
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.