A distinctive property of SII is that it is the first cortical stage of the somatosensory projection pathway that integrates information arising from both sides of the body. However, there is very little known about how inputs across the body mid-line are processed within SII.Optical intrinsic signal imaging was used to evaluate the response of primary somatosensory cortex (SI and SII in the same hemisphere) to 25 Hz sinusoidal vertical skin displacement stimulation ("skin flutter") applied contralaterally, ipsilaterally, and bilaterally to the central pads of the forepaws. A localized increase in absorbance in both SI and SII was evoked by both contralateral and bilateral flutter stimulation. Ipsilateral flutter stimulation evoked a localized increase in absorbance in SII, but not in SI. The SII region that responded with an increase in absorbance to ipsilateral stimulation was posterior to the region in which absorbance increased maximally in response to stimulation of the contralateral central pad. Additionally, in the posterior SII region that responded maximally to ipsilateral stimulation of the central pad, bilateral central pad stimulation approximated a linear summation of the SII responses to independent stimulation of the contralateral and ipsilateral central pads. Conversely, in anterior SII , bilateral stimulation was consistently less than the response evoked from the contralateral central pad.The results indicate that two regions located at neighboring, but distinctly different A-P levels of the anterior ectosylvian gyrus process input from opposite sides of the body midline in very different ways. The results suggest that the SII cortex, in the cat, can be subdivided into at least two functionally distinct regions and that these functionally distinct regions demonstrate a laterality preference within SII. There is general agreement that in cats and monkeys (and presumably in humans) the spike discharge activity a mechanical stimulus sets up in rapidly adapting (RA), slowly adapting (SA), and Pacinian (PC) skin mechanoreceptors is projected centrally, at short latency and with relatively minor transformation, to primary somatosensory cortex (both SI and SII) in the contralateral hemisphere. There is no consensus, however, about the way in which the stimulus-evoked response in the ipsilateral hemisphere contributes to cerebral cortical somatosensory information processing and somatosensation. A distinctive property of SII is that it is the first cortical stage of the somatosensory projection pathway that integrates information arising from both sides of the body. Similar to SI, SII possesses a clear topographic organization , but unlFigure To more accurately characterize the spatial disparity between SII loci activated by contralateral vs. ipsilateral stimulation, the absorbance values were obtained and plotted along the posterior-anterior axis of SII. Figure A more comprehensive view of the SII response to the contralateral and bilateral stimulus conditions can be better appreciated with a multi-dimensional surface plot of the data. Figure To directly compare the time course of the response of the anterior and posterior regions of SII to the three stimulus conditions, we determined the time course of the absorbance changes in each region under each stimulus condition Figure . The ploCluster plots were used to directly compare the response of SII to the different conditions of ipsilateral and contralateral stimulation. In each plot in Figure 2 boxel surrounding the peak. In anterior SII, the majority of the responses to contralateral stimulation are stronger than the responses to bilateral stimulation – hence, the majority of the points plotted fall below the reference line (which has a slope of 1). In the posterior region, the majority of the points plotted are above the reference line – indicating that the response to the bilateral stimulus was greater than the response to the contralateral stimulus.The contralateral and bilateral stimulus conditions can also be compared using cluster analysis. However, because the difference between the contralateral and ipsilateral responses is more robust than the difference between the contralateral and bilateral responses, Figure Thus far, the results suggest that the anterior and posterior regions of SII are differentially activated by contralateral, ipsilateral and bilateral stimulation. To determine the across-subject consistency of these findings, the average absorbance values evoked by the 3 different stimulus conditions were determined for all 6 of the subjects Figure . ClearlyThe findings of this study demonstrated clearly that the anterior and posterior regions of SII process bilateral inputs very differently. At the locus of the maximal OIS response evoked in the posterior region by an ipsilateral stimulus, bilateral stimulation evoked a response that was, on average, about 25% larger than that evoked from the contralateral stimulus site. Conversely, at the locus of the maximal OIS response evoked by contralateral stimulation in the anterior region, bilateral stimulation evoked a response that was, on average, 35% lower than the activity evoked by a contralateral stimulus. This discrepancy between the optical responses of the anterior and posterior regions could be related to neurophysiological observations reported in earlier studies. For example, Carreras and Andersson  found inThe main, although not the only, route for ipsilateral input to SII is through the corpus callosum, from cells located in SI and SII of the opposite cerebral hemisphere ,7,8,11. The neurons in the distal limb regions of SII do receive substantial callosal connections, but these neurons have been reported to lack ipsilateral RFs , indicatA number of interactions between stimuli applied to both hands have been demonstrated in human psychophysical studies. Gilson  found thA recent report  demonstrOne question that the results suggest is whether or not SII can be segregated by laterality preference, in a manner similar to that observed in other sensory systems. Laterality has been demonstrated in the primary sensory cortex of both the visual system and the auditory system of both primates and cats, and the data in this report strongly suggest that there are cortical areas within SII that exhibit preference to ipsilateral or contralateral inputs. In terms of processing information from simultaneous contralateral and ipsilateral stimuli, there could be further similarities between the somatosensory, auditory and visual systems that have yet to be described. Future investigations will aim to further clarify the role of SII in integration of information from inputs across the body midline.The responses evoked by contralateral and ipsilateral flutter stimulation of the central pad of the cat forepaw define functional subdivisions in SII: the two modes of stimulation maximally activate cortical regions that are anterior and posterior to one another, respectively. Bilateral stimulation, or providing simultaneous contralateral and ipsilateral stimulation, reveals, additionally, that the two adjacent cortical areas process bilateral inputs differently. In the posterior region, where ipsilateral stimulation evokes a maximal response, bilateral stimuli evoke a response that is greater than the response evoked by either the individual ipsilateral or contralateral response. In the anterior region of SII, where the contralateral stimulus evokes a maximal response, bilateral stimuli evoke responses that are smaller in magnitude than the responses evoked by the contralateral stimulus.Adult cats  were subjects. All surgical procedures were carried out under deep general anesthesia . After induction of general anesthesia the trachea was intubated with a soft tube and a polyethylene cannula was inserted in the femoral vein to allow administration of drugs and fluids (5% dextrose and 0.9% NaCl). For each subject, a 1.5 cm diameter opening was made in the skull overlying somatosensory cortex, a chamber was mounted to the skull over the opening with dental acrylic, and the dura overlying anterior parietal cortex was incised and removed. Following the completion of the surgical procedures all wound margins were infiltrated with long-lasting local anesthetic, the skin and muscle incisions were closed with sutures, and each surgical site outside the recording chamber was covered with a bandage held in place by adhesive tape.2 between 3.0 – 4.0%; EEG and autonomic signs  were monitored and titrated (by adjustments in the anesthetic gas mixture) to maintain levels consistent with light general anesthesia. Rectal temperature was maintained (using a heating pad) at 37.5°C.Subjects were immobilized with Norcuron and ventilated with a gas mixture  delivered via a positive pressure respirator 1–3 hours prior to the data acquisition phase of the OIS imaging experiments. Respirator rate and volume were adjusted to maintain end-tidal COEuthanasia was achieved by intravenous injection of pentobarbital (45 mg/kg) and by intracardial perfusion with saline followed by fixative . Following perfusion fiducial marks were placed to guide removal, blocking, and subsequent histological sectioning of the cortical region studied. All procedures were reviewed and approved in advance by an institutional committee and are in full compliance with current NIH policy on animal welfare.Results were obtained during stimulation of the contralateral central pad of the forepaw and/or the ipsilateral central pad of the forepaw. The stimuli always consisted of sinusoidal vertical skin displacements  and were applied using a servocontrolled transducer  that is capable of delivering sinusoidal stimuli in the range of 1–250 Hz at amplitudes in the range of 0–1000 microns. The stimuli were delivered independently to the ipsilateral and contralateral skin sites, and also were applied simultaneously to both sites . The stimulus probes were positioned 500 microns beyond the point at which skin contact was detected (via force transducer on the Cantek). The bilateral stimulus protocols reported in this paper were synchronized to start and stop at the same time. The contralateral, ipsilateral and bilateral stimuli were interleaved on a trial-by-trial basis. This approach was used to control for temporal changes in cortical "state" unrelated to stimulus conditions which, if unrecognized, might obscure or modify any differences between the optical responses evoked by the contralateral, ipsilateral and bilateral stimulus conditions.Near-infrared  OIS imaging was carried out using an oil-filled chamber capped with an optical window . Images At the conclusion of the experiment, the imaged cortical region was removed immediately following intracardial perfusion with saline and fixative. The region then was blocked, postfixed, cryoprotected, frozen, sectioned serially at 30 μm, and the sections stained with cresyl fast violet. The boundaries between adjacent cytoarchitectonic areas were identified by scanning individual sagittal sections separated by no more than 300 μm and were plotted at high resolution using a microscope with a drawing tube attachment. The resulting plots then were used to reconstruct a two-dimensional surface map of the cytoarchitectonic boundaries within the region studied with optical and neurophysiological recording methods. The locations of microelectrode tracks and electrolytic lesions evident in the histological sections were projected radially to the pial surface and transferred to the map of cytoarchitectonic boundaries reconstructed from the same sections. As the final step, the cytoarchitectonic boundaries  identified in each brain were mapped onto the images of the stimulus-evoked intrinsic signal obtained from the same subject, using fiducial points (made by postmortem applications of india ink or needle stabs) as well as morphological landmarks . Locations of cytoarchitectonic boundaries were identified using established criteria .A-P = anterior-posteriorRA = rapidly adaptingSA = slowly adaptingPC = PacinianRF = receptive fieldOIS = optical intrinsic signalIR = near infraredEEG = electro-encephalogramMEG = magneto-encephalogramBW and OF participated in the design of the experiments, the data collection, and drafting of the manuscript. SS, JC, and VT made significant contributions to the data collection and the analysis of the data. MT played a major role in all aspects of the development of the manuscript.


Previous studies have shown that spatio-tactile acuity is influenced by the clarity of the cortical response in primary somatosensory cortex (SI). Stimulus characteristics such as frequency, amplitude, and location of tactile stimuli presented to the skin have been shown to have a significant effect on the response in SI. The present study observes the effect of changing stimulus parameters of 25 Hz sinusoidal vertical skin displacement stimulation ("flutter") on a human subject's ability to discriminate between two adjacent or near-adjacent skin sites. Based on results obtained from recent neurophysiological studies of the SI response to different conditions of vibrotactile stimulation, we predicted that the addition of 200 Hz vibration to the same site that a two-point flutter stimulus was delivered on the skin would improve a subject's spatio-tactile acuity over that measured with flutter alone. Additionally, similar neurophysiological studies predict that the presence of either a 25 Hz flutter or 200 Hz vibration stimulus on the unattended hand  would decrease a subject's ability to discriminate between two points on the skin.improved spatial acuity by 20 to 25%, the two-point limen was not significantly affected by substantial changes in stimulus amplitude (between 100 – 200 μm). In contrast, simultaneous stimulation of the unattended hand , impaired spatial acuity by 20% with flutter stimulation and by 30% with vibration stimulation.A Bekesy tracking method was employed to track a subject's ability to discriminate between two-point stimuli delivered to the skin. The distance between the two points of stimulation was varied on a trial-by-trial basis, and several different stimulus conditions were examined: (1) The "control" condition, in which 25 Hz flutter stimuli were delivered simultaneously to the two points on the skin of the attended hand, (2) the "complex" condition, in which a combination of 25 Hz flutter and 200 Hz vibration stimuli were delivered to the two points on the attended hand, and (3) a "bilateral" condition, in which 25 Hz flutter was delivered to the two points on the attended hand and a second stimulus (either flutter or vibration) was delivered to the unattended hand. The two-point limen was reduced  under the complex stimulus condition when compared to the control stimulus condition. Specifically, whereas adding vibration to the unilateral two-point flutter stimulus It was found that the addition of 200 Hz vibration to a two-point 25 Hz flutter stimulus significantly improved a subject's ability to discriminate between two points on the skin. Since previous studies showed that 200 Hz vibration preferentially evokes activity in cortical area SII and reduces or inhibits the spatial extent of activity in SI in the same hemisphere, the findings in this paper raise the possibility that although SI activity plays a major role in two-point discrimination on the skin, influences relayed to SI from SII in the same hemisphere may contribute importantly to SI's ability to differentially respond to stimuli applied to closely spaced skin points on the same side of the body midline. Recently, we reported the development of a semi-automated method for measuring a human subject's ability to discriminate between two points on the skin . In thatimprove a subject's ability to discriminate between two points. Alternatively, recent findings comparing the SI activity evoked by different conditions of contralateral, ipsilateral and bilateral stimulation in the cat show that the magnitude of response in SI evoked by contralateral stimulation is reduced in the presence of an ipsilateral stimulus [Mountcastle and Darian-Smith  proposedstimulus . Similarimproved with same-site vibration and degraded with the addition of either a flutter or vibration stimulus on the opposite, unattended hand.Bekesy tracking algorithms were used to find a subject's two-point limen at the dorsal surface of the right hand under four different stimulus conditions. Exemplary results for a single session (four runs) of a subject are shown in Figure reduced  for the complex condition – the two-point limen tracks at approximately 80% of the values measured under the flutter condition. In contrast, the two-point limen was larger  for both bilateral conditions. In the case in which the opposite or unattended hand was presented with a simultaneous 25 Hz flutter stimulus, the two-point limen tracks approximately 20% higher than the control (attended hand only) condition. Similarly, applying a 200 Hz vibration stimulus simultaneously to the unattended hand resulted in two-point limen values that were approximately 30% higher than the control condition.To determine subject consistency of the above findings, the tracking data collected under each condition for an individual subject were averaged. The data were normalized to the flutter condition since the primary objective of this study was to determine the effect of vibration on the response normally evoked by two-point flutter stimulation. Thus, the two-point limen for the flutter condition was defined as the value "1" and all other distances are plotted as a proportion of the values obtained under the flutter condition [To determine the across-subject consistency of the above findings, the data normalization process applied to the single subject case, as shown in Figure In order to more directly compare the responses measured under each of the stimulus conditions, the tracking values obtained from the last five trials across all subjects was averaged and normalized to the flutter condition Figure . Again, To ensure that the enhanced acuity of a subject under the complex stimulus condition was not due simply to the increased amplitude that resulted from adding vibration to a flutter stimulus (which resulted in a stimulus amplitude of 120 μm), the two-point limen was tracked on the attended hand at 25 Hz flutter of varying amplitudes. Specifically, a separate series of sessions were conducted to track and compare the two-point limen for the amplitudes of 100, 150, and 200 μm in the flutter-only condition. The results were normalized to the distances observed under the 100 μm condition and were plotted in the same manner as the previous results , the two-point limen was not affected. Simultaneous stimulation of the hand contralateral to the attended site, however, impaired or reduced spatial acuity by 20% with a flutter stimulus and 30% with a vibratory stimulus.In the present study, we observed stimulus-dependent effects on two-point tracking of a flutter stimulus at the dorsal surface of the attended hand. The two-point limen was reduced  with a complex stimulus that consisted of 25 Hz flutter and 200 Hz vibration components. Specifically, it was found that adding vibration to the unilateral two-point flutter stimulus Vega-Bermudez and Johnson , using gSummers and Chanter  reportedTommerdahl et al.  comparedThe finding in previous OIS imaging experiments in cats that high-frequency skin stimulation is accompanied by a contralateral absorbance increase in area SII and, simultaneously, by a decline in absorbance in SI in the same hemisphere led Tommerdahl et al.  to consiA recent report described that the ability to localize a stimulus on the fingertips of one hand may be impaired with the interference of a similar stimulus on a fingertip of the opposite hand , suggestIn a previously published report, Vierck and Jones  found thWhen stimuli consisting of two points are oscillated on the skin at low-frequency 25 Hz flutter at distant sites, the peaks of SI response are distinct and non-overlapping Figure . Thus, tIn this paper, we propose a model that predicts a correlation between SI cortical activity and spatial acuity. Spatial acuity, as measured by the two-point limen, can be modified by changing stimulus conditions that would be predicted to have an impact on the SI cortical response. In particular, while vibration has the effect of reducing the spatial extent of SI cortical response normally evoked by flutter, such as when a vibrotactile stimulus comprised of both flutter and vibration is delivered to the skin, it also has the effect of improving a subject's ability to discriminate between two points on the skin. Presumably, this occurs as a result of vibration decreasing the spatial extent of the SI cortical response. Alternatively, stimulus conditions that are known to reduce the magnitude of the SI cortical response without changing the shape of response, such as when a second and simultaneous stimulus is delivered to a homotopic skin site on the opposite unattended hand, result in a reduction in spatial discrimination. While SI is regarded as playing a major role in two-point discrimination, this study provides evidence that other cortical areas that are connected to SI (such as SII) contribute importantly to SI's ability to differentially respond to closely spaced tactile stimuli.Five naïve subjects (21–32 years in age) participated in this psychophysical study. All procedures were reviewed and approved in advance by an institutional review board.Sinusoidal vertical skin displacement stimuli were delivered using the Cantek Metatron CS-525 vertical displacement stimulator . The stimulator made contact with the skin via the two tips of the Two-Point Stimulator (TPS) attachment  fitted to the terminal end of the moving shaft of the stimulator transducer. The TPS is described in detail in a separate report . An adjuThe subject was seated in a chair with arms placed comfortably on a table surface. Both arms were placed on X-ray bags filled with glass beads. The investigators molded the bags to fit the contours of the subject's arms, and when the subject was comfortable and the arms positioned appropriately to allow unimpeded access of the stimulator to the center of the dorsal surfaces of each hand, the bags were made rigid by evacuating them of air (achieved by connecting the bag to a vacuum line). In this way the arms were maintained in a comfortable but stable position for the full duration of the experimental session. The subject was unable to see either the experimenter or the stimulator and stimulus-control instrumentation. White noise presented via headphones eliminated potential auditory cues. A micrometer permitted the stimulator transducers and probe assembly to be lowered towards the predefined skin sites. The micrometer position at which the digital display on the stimulator controllers registered a 0.1–0.2 g change in resistive force was interpreted as the point at which the stimulator probes made initial contact with the skin.A tracking protocol was used to conduct a two-point limen test, which determines the "least two-point separation at which the subject feels (has the subjective impression of) two points,"  at the dVT conducted the experiments, analyzed the data and drafted the manuscript. RD had a role in the conduct and design of the experiments. MT was involved with the design of the experiments and the preparation of the manuscript.


While SII cortex is considered to be the first cortical stage of the pathway that integrates tactile information arising from both sides of the body, SI cortex is generally not considered as a region in which neuronal response is modulated by simultaneous stimulation of bilateral (and mirror-image) skin sites.Optical intrinsic signal imaging was used to evaluate the response of SI and SII in the same hemisphere to 25 Hz sinusoidal vertical skin displacement stimulation ("skin flutter") applied contralaterally, ipsilaterally, and bilaterally (simultaneously) to the central pads of the forepaws. A localized increase in absorbance in both SI and SII occurred in response to both contralateral and bilateral flutter stimulation. Ipsilateral flutter stimulation evoked a localized increase in absorbance in SII, but little or no change in SI absorbance. In the forepaw representational region of SI, however, bilateral stimulation of the central pads evoked a response substantially smaller  than the response to flutter stimulation of the contralateral central pad.The finding that the response of SI cortex to bilateral central pad flutter stimulation is substantially smaller than the response evoked by a contralateral flutter stimulus, together with the recently published observation that a region located posteriorly in SII responds with a substantially larger response to a bilateral flutter stimulus than the response evoked from the contralateral central pad, lead us to propose that the SI activity evoked by contralateral skin stimulation is suppressed/inhibited  by the activity a simultaneous ipsilateral skin stimulus evokes in posterior SII. In contrast, SII has long been known to be activated at short latency by mechanical stimulation of skin sites on both sides of the body midline. Although this differential responsivity of SI and SII to stimulation of contralateral es; e.g. ), the coes; e.g. ,3.Recently, we reported the results from experiments in which we obtained simultaneous observations of the activity evoked in both SI and SII in the same hemisphere of cat cerebral cortex by a 25 Hz sinusoidal vertical skin displacement stimulus ("skin flutter") applied contralaterally, ipsilaterally, or bilaterally to the central pads of the forepaws . Brieflyvs. posterior components that comprise SII cortex in the same hemisphere.This report addresses the response of SI cortex to the same modes of stimulation used in the above-described study that focused on SII . The central finding is that flutter stimulation of the ipsilateral central pad exerts a suppressive/inhibitory influence on the SI response to a 25 Hz flutter stimulus to the contralateral central pad (forepaw). In addition, the temporal relationship between stimulus-evoked activity in selected locations in the responding regions of SI and SII is evaluated quantitatively (using the approach of correlation mapping), revealing a previously unrecognized, and presumably functionally important high degree of coordination between the activities evoked by forepaw stimulation in both SI and the recently identified  anteriorFigure vs. bilateral stimulation at 5 seconds after stimulus onset . The top left panel indicates the orientation of the sampled region of interest (ROI) in SI. The surface plots show the absorbance value at each pixel location in the imaged field, with absorbance defined both by elevation along the z-axis and by pseudocolor . Consistent with the difference images shown in Figure The SI response to each of the different stimulus conditions can be better appreciated when the imaging data are displayed as a multi-dimensional surface plot. Figure vs. contralateral stimulation appears to increase with increasing time after onset of stimulation.Figure vs. bilateral stimulus conditions. Additionally, the plots suggest that there is a time dependency in the development of the response to bilateral stimulation – that is, there is little difference between the responses to the two conditions at 1 sec after stimulus onset , yet at a later time (at t = 5 in each subject many of the pixels are shifted to a position below the reference line). Points located below (or to the right of) the reference line (slope = 1) represent pixels, or spatial locations in SI, where the response to contralateral stimulation is greater than the response to bilateral stimulation. It should be emphasized that this type of graphic does not reflect spatial differences in the responses to the different stimulus conditions, but rather, emphasizes whether or not different members of a set respond differently to the different stimulus conditions. Accordingly, the plots in Figure Scatter plots were used to directly compare the response of SI to the different conditions of contralateral and bilateral stimulation. In each plot in Figure o was that the ratio = 1).The across-subject consistency of the results was evaluated by determining the average absorbance values evoked by the 3 different stimulus conditions (with a 5 sec stimulus duration) for the 5 subjects at 5 sec after stimulus onset . In this method, rather than correlating each pixel with a standard time course observed at one particular cortical region for the same stimulus condition, performs a cross correlates the time courses of absorbance values obtained at the same spatial location under different stimulus conditions. Thus, the correlation coefficient generated at each pixel location represents how similar (indicated by a positive correlation) or how different (indicated by a negative correlation) are the temporal responses of a cortical region recorded under 2 different stimulus conditions. The map in Figure A second method of correlation mapping was used to directly compare the responses evoked by contralateral The findings of this study demonstrate that bilateral flutter stimulation of the central pads of the forepaws evokes an SI response significantly smaller than the response evoked when the stimulus is applied contralaterally. At the locus of the maximal OIS response evoked in the SI region by a contralateral stimulus, bilateral stimulation evoked a response that was, on average, 35% smaller than that evoked by the contralateral stimulus. Concurrently, at the locus of the maximal OIS response evoked by contralateral stimulation in anterior SII , bilateAlthough the neural mechanisms responsible for the above-described effect remains uncertain, some workers have reported that callosally-transmitted inputs exert modulatory, but inconsistent effects on SI neurons. Schnitzler et al , using MAlthough an influence mediated via direct callosal projections from ipsilateral SI to contralateral SI  should nAn extensive literature addresses the neuroanatomical routes by which information about the status of skin mechanoreceptors accesses SI and SII  is negatively correlated with the activity in SI. Additionally, correlation mapping of the results obtained from the contralateral vs. bilateral stimulus conditions demonstrated that the time course of the activities evoked in SI and anterior SII by these conditions are similar. The data lead to the proposal that increasing levels of activity in posterior SII evoked by an ipsilateral skin stimulus suppress/inhibit the responses normally evoked in both anterior SII and SI by contralateral stimulation.Adult cats  were subjects. All surgical procedures were carried out under deep general anesthesia . After induction of general anesthesia the trachea was intubated with a soft tube and a polyethylene cannula was inserted in the femoral vein to allow administration of drugs and fluids (5% dextrose and 0.9% NaCl). For each subject, a 1.5 cm diameter opening was made in the skull overlying somatosensory cortex, a chamber was mounted to the skull over the opening with dental acrylic, and the dura overlying anterior parietal cortex was incised and removed. Following the completion of the surgical procedures all wound margins were infiltrated with long-lasting local anesthetic, the skin and muscle incisions were closed with sutures, and each surgical site outside the recording chamber was covered with a bandage held in place by adhesive tape.Subjects were immobilized with Norcuron and ventilated with a gas mixture  delivered via a positive pressure respirator 1–3 hours prior to the data acquisition phase of the OIS imaging experiments. Respirator rate and volume were adjusted to maintain end-tidal CO2 between 3.0 – 4.0%; EEG and autonomic signs  were monitored and titrated (by adjustments in the anesthetic gas mixture) to maintain levels consistent with light general anesthesia. Rectal temperature was maintained (using a heating pad) at 37.5°C.Euthanasia was achieved by intravenous injection of pentobarbital (45 mg/kg) and by intracardial perfusion with saline followed by fixative . Following perfusion fiducial marks were placed to guide removal, blocking, and subsequent histological sectioning of the cortical region studied. All procedures were reviewed and approved in advance by an institutional committee and are in full compliance with current NIH policy on animal welfare.Results were obtained during stimulation of the contralateral central pad of the forepaw and/or the ipsilateral central pad of the forepaw. The stimuli always consisted of sinusoidal vertical skin displacements  and were applied using a pair of servocontrolled transducers  that is capable of delivering sinusoidal stimuli in the range of 1–250 Hz at amplitudes in the range of 0–1000 microns. The stimuli were delivered independently to the ipsilateral and contralateral skin sites, and also were applied simultaneously to both sites . The stimulus probes were positioned 500 microns beyond the point at which skin contact was detected (via force transducer on the Cantek). The bilateral stimulus protocols reported in this paper were synchronized to start and stop at the same time. The contralateral, ipsilateral and bilateral stimuli were interleaved on a trial-by-trial basis. This approach was used to control for temporal changes in cortical "state" unrelated to stimulus conditions which, if unrecognized, might obscure or modify any differences between the optical responses evoked by the contralateral, ipsilateral and bilateral stimulus conditions.Near-infrared  OIS imaging was carried out using an oil-filled chamber capped with an optical window  Images oij between the absorbance value of each pixel  and the average absorbance value within the reference region over the time from stimulus onset to stimulus offset. The region selected as the reference was defined by a boxel (π mm2 area) centered on the region of interest (ROI). Each pixel  on the correlation map is represented by a coefficient of determination r2ij  for different stimulus conditions. Thus, each coefficient of determination r2ij represents how similar (positive correlation) or how different (negative correlation) the response in a region is to a change in stimulus condition.Correlation maps were constructed for comparison of spatio-temporal characteristics of the OIS response. One aspect of this method of analysis has been previously described in detail ,29. BrieAt the conclusion of the experiment, the imaged cortical region was removed immediately following intracardial perfusion with saline and fixative. The region then was blocked, postfixed, cryoprotected, frozen, sectioned serially at 30 μm, and the sections stained with cresyl fast violet. The boundaries between adjacent cytoarchitectonic areas were identified by scanning individual sagittal sections separated by no more than 300 mm and were plotted at high resolution using a microscope with a drawing tube attachment. The resulting plots then were used to reconstruct a two-dimensional surface map of the cytoarchitectonic boundaries within the region studied with optical and neurophysiological recording methods. The locations of microelectrode tracks and electrolytic lesions evident in the histological sections were projected radially to the pial surface and transferred to the map of cytoarchitectonic boundaries reconstructed from the same sections. As the final step, the cytoarchitectonic boundaries  identified in each brain were mapped onto the images of the stimulus-evoked intrinsic signal obtained from the same subject, using fiducial points (made by postmortem applications of india ink or needle stabs) as well as morphological landmarks . Locations of cytoarchitectonic boundaries were identified using established criteria -38.BW and OF participated in the design of the experiments, the data collection, and drafting of the manuscript. SS and JC made significant contributions to the data collection and the analysis of the data. MT played a major role in all aspects of the development of the manuscript.


It is established that increasing the amplitude of a flutter stimulus increases its perceived intensity. Although many studies have examined this phenomenon with regard to the responding afferent population, the way in which the intensity of a stimulus is coded in primary somatosensory cortex (SI) remains unclear.Optical intrinsic signal (OIS) imaging was used to study the evoked responses in SI of anesthetized squirrel monkeys by 25 Hz sinusoidal vertical skin displacement stimulation. Stimuli were 10 sec duration with a 50 sec inter-stimulus interval. Stimulus amplitude ranged from 50 to 400 microns and different amplitudes were interleaved. Control levels of activity were measured in the absence of stimulation, and used to compare with activation levels evoked by the different stimulus amplitudes. Stimulation of a discrete skin site on the forelimb evoked a prominent increase in absorbance within the forelimb representational region in cytoarchitectonic areas 3b and 1 of the contralateral hemisphere. An increase in stimulus amplitude led to a proportional increase in the magnitude of the absorbance increase in this region of areas 3b and 1 while surrounding cortex underwent a decrease in absorbance. Correlation maps revealed that as stimulus amplitude is increased, the spatial extent of the activated region in SI remains relatively constant, and the activity within this region increases progressively. Additionally, as stimulus amplitude is increased to suprathreshold levels, activity in the surround of the activated SI territory decreases, suggesting an increase in inhibition of neuronal activity within these regions.Increasing the amplitude of a flutter stimulus leads to a proportional increase in absorbance within the forelimb representational region of SI. This most likely reflects an increase in the firing rate of neurons in this region of SI. The relatively constant spatial extent of this stimulus-evoked increase in absorbance suggests that an increase in the amplitude of a 25 Hz skin stimulus does not evoke a larger area of SI neuronal activation due to an amplitude-dependent lateral inhibitory effect that spatially funnels the responding SI neuronal population. The way in which stimulus intensity is represented in primary somatosensory (SI) cortex has remained an intriguing question in the study of the cortical correlates of perception. Although there have been numerous studies of the SI response to changes in stimulus intensity, few have focused on the response at the population level of analysis. Thus, a dearth of information about the global SI response to changes in stimulus intensity exists – current knowledge of the subject depends almost exclusively on reconstruction of predictions of the SI neuronal population response from afferent recordings -5 and si2 [A number of studies have examined the global SI response using imaging techniques such as fMRI  -10 and M2 ,11.Recently, Chen and colleagues used the optical intrinsic signal (OIS) to demonstrate that a proportionally greater (larger magnitude) response is evoked in SI of squirrel monkeys as the amplitude (as measured by force) of a skin stimulus is increased. HoweverFigure To identify the boundaries of the regions of increased absorbance spatial histograms were constructed. Figure Figure evoked was used. ΔAbsorbanceevoked was defined as the difference between the absorbance measured at 1 sec (prior to stimulus onset) and 11 sec (point of stimulus offset), and is shown in the plot at the bottom of Figure evoked vs. amplitude is well described (coefficient of determination R2 = 0.9921) by the linear function (solid line) ΔAbsorbanceevoked = (4 × 10-6)*d + 0.0005. This type of analysis, however, gives little or no information about the spatial properties of the response.The analysis approach illustrated in Figure Radial histograms were constructed to better visualize the spatiotemporal relationship of the OIS response at different amplitudes. The radial histograms shown in Figure max was defined as the difference between the minimum absorbance and the maximum absorbance value obtained at any point during the recording. Interestingly, the relationship between stimulus amplitude and ΔAbsorbancemax in the surround is not linear  to the location of the pixel being compared. This gives a fairly good approximation of the signal at all locations in the image. Since there is no significant difference in the spatial properties between stimuli at intermediate amplitudes  only the 50 and 400 μm amplitudes will be compared with this technique. Figure Correlation maps were constructed to further characterize the spatial properties of the SI response to 25 Hz flutter. A correlation map compares every pixel in the image with the signal referenced from the ROI, and assigns a correlation coefficient (2 (4 × 4) area centered around the ROI. Figure To examine the spatial dynamics of the SI response in more detail we examined the patterns of activity generated by low- and high-amplitude stimulation in a 16 mmThis study evaluated the global SI response to different amplitudes of flutter stimulation by imaging the optical intrinsic signal (OIS). The OIS indirectly reflects both cortical neuronal spike discharge activity and the local, subthreshold changes in neuronal membrane potential evoked by sensory stimulation -16. As a+ and neurotransmitter [One important distinction between previous work done using the OIS and the present study is our use of near-infrared wavelengths for illumination during acquisition of the OIS. The OIS obtained using infrared light has been shown to be highly correlated with light scattering effects that accompany astrocyte swelling subsequent to the clearance of extracellular Knsmitter ,15,18 annsmitter ,20. Althnsmitter  but the nsmitter ,15,19.Previous studies conducted by this laboratory have reported that the SI optical response evoked by an extended period (>1 sec) of flutter stimulation not only consists of an increase in absorbance in the region that receives its input from the skin site that was stimulated, but also decreases in absorbance (frequently to levels well below-background) that occur in the surrounding cortex . The preThe correlation maps shown in figure A receptor mediated inhibition (barbiturates), significantly reduce the dimensions of the receptive field of individual SI neurons; actions that would reduce the size of the responding SI neuronal population [Examination of spatial histograms figure  and the pulation . Chen etpulation .It has been suggested that the amplitude of skin flutter stimulation is coded by both the number of activated SI neurons as well as by their level of spike discharge activity . This suThis study investigated the SI response to flutter stimulation of the skin using the OIS. An increase of the amplitude of the flutter stimulus was associated with an increase in absorbance within the region of SI cortex that receives its input from the stimulated skin field. The relationship between the maximal change in absorbance and stimulus amplitude is well characterized by a linear function within the range of amplitudes studied. Measurement of the spatial extent of the activated SI region showed that higher amplitudes of stimulation do not produce a more extensive region of SI activation. Instead, as amplitude is increased, while average peak absorbance within the same ~2 mm diameter SI region increases with amplitude of stimulation, the region of surrounding cortex undergoes a prominent decrease (frequently to levels well below background) in absorbance. Further studies are required to establish the relationship between the effect of different amplitudes of skin flutter stimulation on SI absorbance and SI neuroelectrical activity.2O) and oxygen, the trachea was intubated. A veterinary anesthesia machine (Forreger Compac-75) provided an anesthetic gas mix whose composition could be adjusted  to maintain a stable level of surgical anesthesia. Methylprednisolone sodium succinate (20 mg/kg) and gentamicin sulfate (2.5 mg/kg) were injected intramuscularly to lessen the probability of halothane-induced cerebral edema and prevent bacterial septicemia, respectively. Placement of a valved catheter into a superficial hindlimb vein enabled administration of 5%glucose, 0.9%NaCl, and drugs.All methods and procedures are consistent with USPHS policies and guidelines on animal care and welfare in biomedical research. They were reviewed and approved by an institutional committee prior to initiation of the experiments. Experiments were conducted in 10 squirrel monkeys. Following induction of anesthesia with 4% halothane in a 50/50 mix of nitrous oxide  to maintain heart rate, blood pressure, and the EEG at values consistent with general anesthesia. Rate and depth of ventilation were adjusted to maintain end-tidal CO2 between 3.0 and 4.5%. Under these experimental/anesthetic conditions both SI neuron spontaneous and stimulus-evoked spike discharge activity patterns are highly reproducible over even prolonged (>1 hr) time periods.A 1.5 cm opening was trephined in the skull overlying SI cortex. A recording chamber (25 mm i.d.) was placed over the opening and cemented to the skull with dental acrylic. Wound margins were infiltrated with local anesthetic, closed with sutures and bandaged, and the dura overlying SI was resected. After the completion of all surgical procedures subjects were immobilized with norcuron . From this point on, the animal was ventilated with a 50/50 mix of NAfter obtaining a photograph of the exposed cortical surface, the recording chamber was filled with artificial cerebrospinal fluid, and hydraulically sealed using a clear glass plate. In each of five experiments, the OIS evoked in SI by cutaneous flutter stimuli on the thenar region of the forelimb was recorded. The flutter stimulus was delivered at 50, 100, 200 and 400 μm and was interleaved in order to prevent conditioning of the response. The imaging system consisted of a computer-interfaced CCD camera (Quantix 540 from Roper Scentific), light source, guide and filters required for near-infrared (833 nm) illumination of the cortical surface, a focusing device, and a recording chamber capped by an optical window . Images of the exposed anterior parietal and surrounding cortical surface were acquired 200 ms before stimulus onset ("reference images") and continuously thereafter for 22 s after stimulus onset ("post-stimulus images") at a rate of one image every 0.9–1.4 s. Exposure time was 200 ms. Difference images were generated by subtracting each pre-stimulus image from its corresponding post-stimulus image. Averaged difference images typically show regions of both increased light absorption (decreased reflectance) and decreased light absorption (increased reflectance) which are believed widely  to be accompanied by increases and decreases in neuronal activation, respectively.Although onset of the OIS is delayed at longer wavelengths ,20, use ij between the reflectance value of each pixel  and the average reflectance value within the reference region over the time from stimulus onset to stimulus offset. The region selected as the reference was defined by a boxel (π mm2 area) centered on the region of interest (ROI). Each pixel  on the correlation map is represented by a correlation coefficient ij r identified in each brain were mapped onto the images of the stimulus-evoked intrinsic signal obtained from the same subject, using fiducial points (made by postmortem applications of india ink or needle stabs) as well as morphological landmarks . Locations of cytoarchitectonic boundaries were identified using established criteria -31.SS participated in acquisition of optical data, performed analysis of the data and drafted the manuscript. VT and JC assisted in the data collection and the analysis of the optical imaging data. OF and BW participated in the design of the study, the conduct of the experiments and the drafting of the manuscript. MT participated in the design, conduct, and analysis of the experiments, and in the manuscript preparation.


Recently we reported that vibrotactile flutter stimulation of a skin locus at different amplitudes evokes an optical response confined to the same local region of the primary somatosensory cortex (SI), where its overall magnitude varies proportionally to the flutter amplitude. In this report, we characterize the impact of the flutter amplitude on the spatial patterns of activity evoked within the responding SI region.In order to characterize the spatial pattern of activity within the responding SI region, images of the flutter-evoked SI optical response were segmented and analyzed with spatial frequency analysis. The analysis revealed that: (1) dominant spatial frequencies in the optical intrinsic signal emerge within the responding SI region within 3–5 sec of stimulus onset; (2) the stimulus-evoked activity is spatially organized in a form of several roughly parallel, anterior-posteriorly extended waves, spaced 0.4–0.5 mm apart; (3) the waves themselves exhibit spatial periodicities along their long axis; and (4) depending on the flutter stimulus amplitude, these periodicities can range from fine 0.15 mm "ripples" at 50 μm amplitude to well-developed 0.5 mm fluctuations at the amplitude of 400 μm.The observed spatiointensive fractionation on a sub-macrocolumnar scale of the SI response to skin stimulation might be the product of local competitive interactions within the stimulus-activated SI region and may be a feature that could yield novel insights into the functional interactions that take place in SI cortex. In this map, a skin locus provides afferent input to an extensive cortical region in SI ,2. In pacortex – ,4). The cortex – . These ccortex – -10.2 of cortical territory in area 3b of SI [Such spatially extensive cortical regions are not functionally homogeneous. For example, using Optical Intrinsic Signal (OIS) imaging in near-infrared 830 nm) range, we find that in squirrel monkeys a small-diameter stimulus probe oscillating on the skin at 25 Hz activates more than 3 mm3b of SI ,8,11. Su30 nm ran3b of SI ) organiz3b of SI . Chen et3b of SI  reported3b of SI ,14-18.Together these considerations suggest that the spatial pattern of activity evoked in SI by even the smallest stimuli might be structurally more complex than a typically envisioned basic bell-shaped pattern. A closer inspection of such patterns might reveal certain spatial formations within them with significant functional implications. Recently, we investigated the response of SI cortex to varying amplitudes of flutter stimulation. Regardless of the amplitude of stimulation (in the range of 50 to 400 μm), we found that the spatial extent of the response of SI cortex remained the same . InsteadFigure Comparable results were obtained from the 4 other subjects,  of mechanical skin stimulation. Similarly, Bruno and colleagues recently demonstrated that individual barrels in rat SI contain minicolumns of neurons preferring the same whisker deflection angle and that these angular tuning domains could be the result of convergent inputs from thalamocortical cells with corresponding angular preferences . It is pFinally, considering that spatial periodicities in SI OIS response patterns emerge gradually, and evolve with time after the stimulus onset, the fine sculpting of the SI response might be the result of a network-level neurocomputational process that involves competitive and cooperative interactions among local neuronal aggregates. That is, SI sensitivity to the stimulus attribute(s) responsible for the observed fractionation of activity within the responding SI region might be an emergent property of the SI network , rather than a simple outcome of selective convergence of thalamocortical afferents on SI neurons.Observations of the spatial patterns of SI cortical response within an activated region, such as those evoked by flutter stimulation of the skin, suggest that evoked cortical activity within such a territory is not evenly distributed. Furthermore, the cortical activity patterns change in a manner that appears to be dependent upon stimulus conditions. The observed spatiointensive fractionation on a sub-macrocolumnar scale of the SI response to skin stimulation might be the product of local competitive interactions within the stimulus-activated SI region, and as such can lead to new insights about the functional interactions that take place in the SI cortex.All methods and procedures are consistent with USPHS policies and guidelines on animal care and welfare in biomedical research, and were reviewed and approved in advance by an institutional animal use committee (IACUC). Experiments were conducted in 5 squirrel monkeys. Surgical procedures were carried out under deep general anesthesia . After induction of anesthesia the trachea was intubated to facilitate positive pressure ventilation and delivery of the gaseous general anesthetic. A catheter was inserted into a branch of the femoral vein of the hindlimb ipsilateral to the hemisphere to be imaged, allowing intravenous (IV) administration of drugs and fluids (5% dextrose and 0.9% NaCl). Methylprednisolone sodium succinate (20 mg/kg) and gentamicin sulfate (2.5 mg/kg) were injected intramuscularly to lessen the probability of halothane-induced cerebral edema and prevent bacterial septicemia, respectively.A 1.5 cm diameter opening in the skull exposed the forelimb region of SI cortex. A recording chamber was positioned over the opening and cemented to the skull with dental acrylic. The chamber was filled with artificial cerebrospinal fluid, the dura mater overlying SI cortex incised and removed, and all wound margins outside the chamber dressed with long-lasting local anesthetic in oil (Cetacaine). All skin and muscle incisions were closed with sutures and bandaged.After the completion of all surgical procedures subjects were immobilized with Norcuron  and ventilated with a gas mixture . At these concentrations and under normocapnic conditions, halothane has no effect on brain energy metabolism ,37, and The recording chamber was filled with artificial cerebrospinal fluid and hydraulically sealed using a clear glass plate. Vibrotactile stimuli were delivered to selected loci on the hand using a servocontrolled vibrotactile stimulator , capableThe optical imaging system consisted of a computer-interfaced CCD camera, light source, guide and filters required for near-infrared (830 nm) illumination of the cortical surface, a focusing device, and a recording chamber capped by an optical window . ImaCortical images were taken at light/time exposures that place the region of interest in the middle of the range of the recorded pixel values. Histogram analysis was used during experimental setup to make sure to avoid any nonlinearities that may arise from overexposure. In some of the experiments the camera was rotated by 90° relative to the SI orientation to better capture the responding cortical field. These rotations did not have any noticeable effect on spectral power distributions along the anterior-posterior and medial-lateral cortical dimensions.vs. distance plot . Power spectra of the spatial histograms were then computed using Discrete Fourier Transform (DFT) algorithm and plotted as a periodogram. Fourier analysis was always performed on raw, unfiltered images.The spatial organization of the stimulus-related light absorbance changes in SI was evaluated using linear image segmentation. This involved segmentation of the relevant region of the image into a linear series of bins and computation of the average absorbance value of the pixels in each bin. The sequence of average absorbance values obtained in this way was plotted as a function of distance (mm) along the cortical path traced by the central points of the series of bins – yielding an absorbance At the end of the experiment the subject was euthanized by overdose of pentobarbital (50 mg/kg/IV), followed by intracardial perfusion with saline and 10% formalin.JC participated in acquisition of optical data, performed analysis of the data and drafted the manuscript. OF and BW participated in the design of the study, the conduct of the experiments and the drafting of the manuscript. MT participated in the design, conduct, and analysis of the experiments, and in the manuscript preparation.


To compare the diagnostic accuracy of two continuous screening tests, a common approach is to test the difference between the areas under the receiver operating characteristic (ROC) curves. After study participants are screened with both screening tests, the disease status is determined as accurately as possible, either by an invasive, sensitive and specific secondary test, or by a less invasive, but less sensitive approach. For most participants, disease status is approximated through the less sensitive approach. The invasive test must be limited to the fraction of the participants whose results on either or both screening tests exceed a threshold of suspicion, or who develop signs and symptoms of the disease after the initial screening tests.paired screening trial bias. This bias reflects the synergistic effects of inappropriate reference standard bias, differential verification bias, and partial verification bias. The absence of a gold reference standard leads to inappropriate reference standard bias. When different reference standards are used to ascertain disease status, it creates differential verification bias. When only suspicious screening test scores trigger a sensitive and specific secondary test, the result is a form of partial verification bias.The limitations of this study design lead to a bias in the ROC curves we call paired screening trial bias on a paired comparison of area under the curves. We fix the prevalence of disease, and the chance a diseased subject manifests signs and symptoms. We derive the formulas for true sensitivity and specificity, and those for the sensitivity and specificity observed by the study investigator.For paired screening tests with bivariate normally distributed scores, we give formulae and programs to quantify the effect of The observed area under the ROC curves is quite different from the true area under the ROC curves. The typical direction of the bias is a strong inflation in sensitivity, paired with a concomitant slight deflation of specificity.In paired trials of screening tests, when area under the ROC curve is used as the metric, bias may lead researchers to make the wrong decision as to which screening test is better. Paired trials designed to compare the diagnostic accuracy of screening tests using area under the receiver operating characteristic (ROC) curve may fall victim to a strong bias that renders the conclusions of the trial incorrect. In English, "bias" often has a pejorative connotation, implying that those who conduct the study prefer one scientific conclusion, rather than another. We use the term "bias" in the epidemiological and statistical sense, as the difference between the results obtained in a study, and the true results.The bias occurs because limitations in the trial design may differentially affect the area under the ROC curve for each screening test. Many competing statistical approaches have been suggested for comparing the diagnostic accuracy of two continuous tests. We consider area under the ROC curve, because it continues to be used as the standard in prominent medical journals -3.true disease status is not known correctly for all participants, the observed disease status is used for calculations of diagnostic accuracy.A common design for the comparison of two continuous screening tests is to evaluate participants with both screening tests. The disease status is then determined by either an invasive secondary test, or by a less invasive, but less sensitive approach. Ethically and practically, the invasive secondary test must be reserved only for those participants who have a suspicious result on one or both screening tests, or for those who have signs and symptoms of disease. For those who have a normal result on both screening tests, a less sensitive process is used to approximate the disease status. As the For potentially lethal diseases like cancer, where the invasive test is biopsy, this design is the best possible available design. The imperfections of the study design occur because the disease is difficult to diagnose since it is clinically occult, and the study designers must keep the risk of potential harm to subjects as low as possible.paired screening trial bias. This bias results from the synergistic effects of inappropriate reference standard bias, differential verification bias, and partial verification bias , standard deviations both 1 and correlation ρ, evaluated at the points x and y. That is, if X and Y have a bivariate normal distribution, we write Φ to indicate Pr (X ≤ x and Y ≤ y|ρ)The prevalence of disease in the population is θ, the threshold value for referral to the invasive, yet sensitive and specific secondary test. Thus, for each value of x, we can describe a series of events cross-classified by the Test 1 score, the Test 2 score, the true disease status of the subject and the presence of signs or symptoms. We classify each event both as it truly occurs, and how it is observed by the study investigator. There are 22 possible situations when x <θ , and the truth (the presence or absence of disease). The cell and marginal probabilities for this cross-classification are shown in Tables For each screening test and each value of the test cutpoint true sensitivity for each test as the number of true positives identified by that screening test divided by the total number of true cases. The true specificity for each test is the number of true negatives correctly identified as negative by that screening test divided by the total number of true non-cases. The true ROC curve is generated by plotting the true sensitivity on the vertical axis versus one minus the true specificity on the horizontal axis.We then calculate the observed sensitivity and observed specificity. In order to generate the observed ROC curves, for each test and each value of the cutpoint x, we define a table that cross classifies the response of the test (positive or negative), and the observed disease status (the presence or absence of observed disease). The cell and marginal probabilities for this cross-classification are shown in Tables observed sensitivity and the observed specificity. The observed sensitivity is the fraction of participants observed to have disease who have a positive screening test result. The observed specificity is the fraction of participants who apparently have no disease who have a negative screening test result. Some participants actually may have disease, but the disease is not detected in the trial.We use a similar technique to calculate the observed ROC curve is generated by plotting the observed sensitivity on the vertical axis versus one minus the observed specificity on the horizontal axis. Simpson's rule numerical integration methods We calculate the theoretically correct ROC curves and AUCs (ignoring the error of integration), using our mathematical derivations. In a real trial, the study investigator would use a hypothesis test and a p-value to compare the difference in AUCs. Depending on the sample size chosen for the trial, the precision of the estimates and the accuracy of the decision may change.true state of disease, and the observed state of disease, we record the magnitude of the differences in AUCs, and the decision whether to reject the null. The proportion of rejections for the true and observed data is estimated by the number of rejections, divided by 10,000. Ten thousand is chosen so the maximum half width for the confidence interval for the proportion rejected is no more than 0.01.To illustrate the effect of the bias, we present the theoretical results. To illustrate the effect of sample size on the precision of the estimates, we conduct a simulation. For the simulation, we suppose that the study investigator decided to test the null hypothesis of no difference between the areas under the ROC curves, using a non-parametric AUC test for paired data , and fixobserved ROC curve differs from the true ROC curve, with the amount of bias depending on the correlation between the screening tests for participants with disease, ρC, the rate of signs and symptoms, ψ, and the threshold for recall, θ. In some cases, the bias equally affects the observed ROC curve for both screening tests, and the scientific conclusion is the same as it would have been had the true disease state been observed. In other cases, the bias causes a change in the direction of the scientific conclusion. The scientific conclusion only changes direction when for one screening test, for participants with disease, a higher proportion of the scores lead to recall than for the other screening test. Thus, for that screening test, a larger percent of participants with true disease go on to have their disease status correctly ascertained, and observed in the study, than for the other screening test.Our derivations demonstrate that the Figure observed curves have inflection points, where the slope changes. There is no inflection point in the true ROC curves for either test, because the formulae that govern the sensitivity and specificity for the true curves are the same no matter what the ROC cutoff points are . However, in Figure observed difference in AUC between Test 2 and Test 1 is negative (Test 2 observed AUC – Test 1 observed AUC = 0.75 – 0.82 = -0.07). If the study investigator were to observe these exact theoretical results, the study investigator would conclude that screening Test 1 has better diagnostic accuracy than Test 2, when in fact the opposite is true.Since Test 2 truly has better diagnostic accuracy than Test 1, the true and observed disease status, we conducted a simulation. For the parameters of Figure Study investigators never observe the true state of nature. They observe data, and make estimates, the precision of which depends on the sample size. They decide which screening test is better using hypothesis tests. To see which conclusion the hypothesis tests would suggest, both for the observed state of disease. In that case, the study investigator will reject the null hypothesis only 71% of the time. The remaining 29% of the time, the study investigator will conclude that there is no difference in AUC between Test 1 and Test 2. The lower power is due to more variance in the observed data, compared to the true data. When the study investigator rejects the null, every time, she concludes incorrectly that Test 1 is better than Test 2.If we conduct the same simulation experiment from the point of view of the study investigator, for the experimental situation of Figure observed sensitivity for Test 1 is inflated more than the observed sensitivity for Test 2. The increase in observed sensitivity makes the observed ROC curve higher for Test 1 than for Test 2. A higher observed ROC curve means a higher observed AUC for Test 1 than for Test 2.The incorrect conclusion in Figure true and observed ROC curves shown in Figure observed sensitivity relative to true sensitivity, for Test 1. This occurs when specificity is 0.82. For a hypothetical study of 10,000 participants, and specificity of 0.82, the observed and true 2 × 2 tables for Test 1 and Test 2 are shown in Figure To understand how and why paired screening trial bias occurs, consider a single specificity value on the observed table, Test 1 is positive if it exceeds 2.511; for the true table, Test 1 is positive if it exceeds 2.515. For the observed table, Test 2 is positive if it exceeds 1.269; for the true table, Test 2 is positive if it exceeds 1.265. The tables have different ROC cutpoints because they were chosen to have the same specificity, not the same cutpoint.Each one of the four tables uses a slightly different ROC cutpoint. For the observed number of cases of disease is smaller than the true number because not every participant undergoes the invasive, yet sensitive and specific secondary test, and thus some cases of disease are missed. The observed number of cases of disease is the denominator of the observed sensitivity. Because the denominator is smaller for observed sensitivity than for true sensitivity, the observed sensitivity is strongly inflated for both tests. When specificity is 0.82, the observed sensitivity of Test 1 is 0.72, with true sensitivity of 0.33. For Test 2, the observed sensitivity is 0.52, with true sensitivity of 0.43.Also, the number of cases of disease observed in the study, 45, is much smaller than the true number of cases of disease in the population, 100. The Yet if the bias only affected the denominator, the inflation in sensitivity would be the same for both tests. After all, the same number of observed cases is used as the denominator for both tests. The differential inflation for Test 1 compared to that for Test 2 must be due to the numerator of the observed sensitivity.For Test 2, the numerator of the observed sensitivity is the number of study participants who are positive on Test 2, and who are observed to have disease in the study. For Test 2, the numerator for observed sensitivity, 23, is smaller than the true numerator, 43. The difference occurs because disease can only be observed if the invasive, yet sensitive and specific secondary test is used. Even though the participants have a score that exceeds the ROC cutpoint for Test 2, they do not all undergo the invasive, yet sensitive and specific secondary test. Thus, they do not yield observed cases of disease. By contrast, for Test 1, because the ROC cutpoint is higher than the threshold which leads to the invasive, yet sensitive and specific secondary test, every participant positive on Test 1 undergoes the secondary test, and is shown to have disease. For each test, there is a different proportion of participants who exceed the cutpoint, who truly have disease, and who proceed to secondary testing. This is the source of the differential bias that causes the curves to reverse order in Figure Paired screening trial bias also increases as the proportion of participants with disease who have signs and symptoms (ψ) decreases. If all the cases of the disease were observed during the trial, there would be no difference between true and observed disease status, and no bias. Yet, in every screening trial, some cases of diseases are not identified by either screening test, and never present with signs and symptoms. As the proportion of participants presenting with signs and symptoms (ψ) decreases, fewer cases of disease are discovered during the trial in the interval after screening, and the difference between observed and true disease status grows.Paired screening trial bias increases with the increase in correlation between the results of the screening tests for participants with disease, ρC. The bias in the observed ROC curves increases because as the two index tests become more highly correlated, the number of observed cases of disease becomes smaller relative to the number of true cases of disease. When the two index tests are highly correlated, they essentially produce the same information as to whether a participant has disease. When the index tests are independent, each test makes diagnoses on its own that the other test misses. Thus, when the tests are independent, and ρC is 0, the number of observed cases is highest, relative to the number of true cases. The percentage of participants receiving the infallible secondary test increases as ρC decreases. The bias lessens as the true disease status is ascertained for more participants.paired screening trial bias tends to strongly increase the sensitivity, while slightly decreasing the estimate of specificity. The increase in observed sensitivity compared to true sensitivity is expected with verification bias [In general, ion bias .In this paper, we define a new type of bias that is a result of the interaction between a particular design for a paired screening trial, and the choice of a particular statistical test. Specifically, the bias occurs when the diagnostic accuracy of two continuous tests are compared using area under the ROC curve in a design with two limitations. First, different methods are used to ascertain disease status, depending on the results of the initial screening tests. Secondly, only some subjects undergo an invasive, yet sensitive and specific secondary test. Thus, some cases of disease are missed because the method used to ascertain disease status for those who test negative on both initial screening tests may not be 100% sensitive.Both the statistical test and the trial design we considered were modeled closely after recently completed and published trials -3. TheseAlthough we modeled our trial design on real trials, we made simplifying assumptions, which may not accurately reflect reality. We assumed that there was a method for determining disease status which was infallible. In reality, all methods of determining disease status may be fallible. In breast cancer, for example, diagnostic mammography, biopsy and follow-up all make errors. Too short a follow-up time may miss cases of disease. While longer follow-up time will reveal a larger fraction of occult disease, it may also reveal increasing numbers of cases of disease that developed after the initial screening period, thus confusing the results. We assumed that all cases of disease are harmful. In screening studies, cases of disease may resolve, or proceed so slowly as to be considered harmless.We assumed that a test to determine disease status would be conducted any time a screening test result exceeded a given threshold. However, in cancer screening, because other factors may be taken into consideration when deciding a course of clinical action, there is a range of scores that may result in further testing.We also made the simplifying assumption that the scores of the screening tests followed a bivariate normal distribution. In real paired cancer trials, the scores have a conditional probability structure driven by the fact that real observers miss cancers (and score a screening test as if no disease were present), and see cancer where there is none . The resulting distribution of scores is far from the bivariate normal distribution we assumed.There is some theoretical justification that our results will still hold even if the data are non-normal. Hanley  points opaired screening trial bias.The previous literature on bias provides some hint of the plethora of possible designs and tests used for statistical analysis. Most previous statistical literature dealt with biases that occur for single, as opposed to paired, tests. A complete summary of biases is given in . Extremepaired screening trial bias, because the secondary test is typically biopsy. Negative screening results cannot lead to biopsy because there is no visible lesion to be biopsied. Because biopsy is painful and invasive, it is infeasible and unethical to do a biopsy unless there are suspicious screening test results. Also, one can only biopsy what one can see: one cannot put a needle in an invisible lesion. Negative screening test results are verified, but typically by follow-up, which has lower sensitivity than biopsy.Cancer screening trials in particular are susceptible to et al., [Our research suggests that in many published paired screening trials, bias did not affect the scientific conclusion. For example, in Pisano et al., , digitalWhy criticize a trial design, that though imperfect, cannot be improved, because of ethical constraints? It is our philosophy that it is preferable to understand all the causes of bias. With mathematical formulae for bias, we can defend trials that are fundamentally correct, and reserve doubt for those trials that may be subject to incorrect conclusions. In addition, models for bias are the necessary first step toward mathematical corrections for bias in sensitivity and specificity, and toward designing new clinical trial methodologies.paired screening trial bias has the potential to subvert the results of paired screening trials, especially when the fraction of the population recalled for secondary testing differs for each screening test. The bias is affected by the rate at which diseased participants experience signs and symptoms of disease, and the chance of recall for a sensitive secondary test. The bias is also influenced by the distributions of the scores for the cases and non-cases for each screening test, and by the correlation between the screening tests. Further research on this bias is needed, so that mathematical corrections for paired screening trial bias can be developed.Using a simplified paradigm, we have shown that Programs implemented in SAS and Mathematica to calculate the true and observed sensitivity, specificity, ROC curves, and areas under the curves are available by request from the authors.ROC: Receiver operating characteristic; AUC: Area under the receiver operating characteristic curve.Financial support for this study was provided by NCI K07CA88811, a grant from the National Cancer Institute to the Colorado School of Public Health, Deborah Glueck, Principal Investigator. The funding agreement ensured the authors' independence in designing the study, interpreting the data, writing, and publishing the report. The authors declare that they have no competing interests.DHG conceived of the idea and derived the mathematics. The first draft of the manuscript was a collaborative effort of DHG and MML. MML provided epidemiological expertise. CIO programmed the formulae and produced graphs. KEM provided advice about the mathematics. BMR and JTB aided in a thorough revision of the manuscript. JML and EDP provided clinical expertise and suggestions for how to relate the topic to medical studies. TAA assisted in the literature review, and in checking the math, and collaborated on the revision of the manuscript. All authors read and approved the final manuscript.For those readers not familiar with ROC analysis, we give a short tutorial. For a complete discussion, see ;Chapter ; ChapterThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/9/4/prepub


The two highly similar Arabidopsis apyrases AtAPY1 and AtAPY2 were previously shown to be involved in plant growth and development, evidently by regulating extracellular ATP signals. The subcellular localization of AtAPY1 was investigated to corroborate an extracellular function.AtAPY1 fused to the SNAP--tag were used for indirect immunofluorescence and AtAPY1 was detected in punctate structures within the cell. The same signal pattern was found in seedlings stably overexpressing AtAPY1-GFP by indirect immunofluorescence and live imaging. In order to identify the nature of the AtAPY1-positive structures, AtAPY1-GFP expressing seedlings were treated with the endocytic marker stain FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)-pyridinium dibromide) and crossed with a transgenic line expressing the trans-Golgi marker Rab E1d. Neither FM4-64 nor Rab E1d co-localized with AtAPY1. However, live imaging of transgenic Arabidopsis lines expressing AtAPY1-GFP and either the fluorescent protein-tagged Golgi marker Membrin 12, Syntaxin of plants 32 or Golgi transport 1 protein homolog showed co-localization. The Golgi localization was confirmed by immunogold labeling of AtAPY1-GFP. There was no indication of extracellular AtAPY1 by indirect immunofluorescence using antibodies against SNAP and GFP, live imaging of AtAPY1-GFP and immunogold labeling of AtAPY1-GFP. Activity assays with AtAPY1-GFP revealed GDP, UDP and IDP as substrates, but neither ATP nor ADP. To determine if AtAPY1 is a soluble or membrane protein, microsomal membranes were isolated and treated with various solubilizing agents. Only SDS and urea  were able to release the AtAPY1 protein from microsomal membranes.Transgenic Arabidopsis lines expressing AtAPY1 is an integral Golgi protein with the substrate specificity typical for Golgi apyrases. It is therefore not likely to regulate extracellular nucleotide signals as previously thought. We propose instead that AtAPY1 exerts its growth and developmental effects by possibly regulating glycosylation reactions in the Golgi. Tagging AtAPY1 was chosen over raising antibodies against it because AtAPY1 and AtAPY2 are so identical in their amino acid (aa) sequence: There is only one six-aa-stretch in AtAPY1 (aa 44–49) that has four different and two similar aa to the corresponding sequence in AtAPY2. All oth6-alkylguanine-DNA alkyltransferase, SNAP binds covalently to benzylguanine-based substrates. There are a large number of substrates coupled to different fluorescent dyes and other labels commercially available making the SNAP-tag a versatile tool for localization studies. The expression of AtAPY1-SNAP was placed under the control of the native promoter region, because overexpression can lead to localization artifacts. Despite this risk, another tagged AtAPY1 version, AtAPY1-GFP, was fused to the strong cauliflower mosaic virus 35S promoter because expression levels of NTPDases are generally low[Among the tags available, the SNAP-tag,45 seemeally low.AtAPY1 and AtAPY2 (DKO) is seedling-lethal[35S promoter made the rescue of the DKO mutant with AtAPY1-GFP impossible as confirmed experimentally, because this promoter is turned off in pollen[apy2 SKO plants  were used as the genetic background for transformation with each tagged AtAPY1 construct. These plants carried AtAPY2 under the control of the pollen-specific promoter SPIK which ensured the survival of the DKO pollen.The SNAP- or GFP-tag was fused to the C-terminus of AtAPY1 to avoid losing the tag by a possible N-terminal cleavage in a subcellular targeting process. Since tags can impair protein function and lead to mislocalization, a complg-lethal. A DKO sn pollen. Withoutn pollen. In ordeAtAPY1::AtAPY1-SNAP or 35S::AtAPY1-GFP and used for genotyping by PCR. Several DKO plants without a WT AtAPY1 and AtAPY2 gene, but with a tagged apyrase construct were identified hereafter called DKO-SNAP and DKO-GFP, respectively. The PCR analysis of two such mutants is shown in FigureSPIK::AtAPY2 construct was always present in the DKO-GFP mutants as expected, but interestingly also in the DKO-SNAP mutants. One possible explanation is that some regulatory elements necessary for optimal expression in pollen were missing in the chosen promoter region. The promoter region used previously for AtAPY1::GUS analyses[AtAPY1. Since the gene At3g04090 was deemed unnecessary for successful complementation, it was mostly excluded in the AtAPY1::AtAPY1-SNAP construct.DNA was isolated from progeny of the partially complemented SKO plants containing either analyses,17,18 wa+/+; apy2/apy2; SPIK::AtAPY2; AtAPY1::AtAPY1-SNAP) and SKO-GFP  plants were included in the study to check for possible dominant negative effects of the tagged apyrase on the WT phenotype. DKO seedlings without a construct coding for a tagged AtAPY1 had an abnormal phenotype with fleshy cotyledons and no root  could be rescued by transformation with AtAPY1-SNAP or AtAPY1-GFP making the DKO-SNAP and DKO-GFP plants suitable tools for localization studies.The lethal DKO . Although specific labeling of fusion proteins in yeast and animin yeast, a high AtAPY1 is expressed strongly in root hairs as well as in guard cells among other cell types[Therefore, indirect immunofluorescence was chosen as a different approach. DKO-SNAP seedlings were fixed. After cell wall digestion and plasma membrane permeabilization, they were incubated with primary antibodies against the SNAP-tag. Following several washing steps, FITC-labeled secondary antibodies were added to visualize AtAPY1-SNAP for the CLSM. The background was low, but no specific signals could be detected (data not shown). To increase the fluorescent signal, the tyramide signal amplification (TSA) technique was applied. This tell types,18.AtAPY1, the indirect immunofluorescence approach was repeated with transgenic plants expressing AtAPY1-GFP under the control of the strong 35S promoter. We used primary antibodies against GFP and secondary Alexa Fluor 488-coupled antibodies in two different approaches: 1. Post-embedding labeling of 200-nm-thin Tokuyasu cryo-sections .The method of live imaging of GFP-tagged proteins is suitable to detect apyrase in the cell wall as shown for apoplastic apyrases in other plant species,54. But d FigureG which dd FigureH, but thd FigureH. This re FigureI, J. Seeapoplast. Howeverin vivo imaging, revealed the same punctate structures, but no signals at the plasma membrane or in the extracellular space.Both detection methods, immunofluorescence and trans-Golgi network, but not the Golgi apparatus[35S::AtAPY1-GFP seedlings did not reveal any co-localization of the fluorescent dye and the GFP signal, even 120 min after the infiltration  and SYP32 (Syntaxin of plants 32) are SNARE proteins localized in the Golgi apparatus. Got1p (erevisiae and its r) of the two fluorescent signals  and 12 nuclei (= 27 μm2), respectively. Multivesicular bodies (MVB) were also positively immunolabeled by gold particles [Acer pseudoplatanus)[If the Golgi localization of AtAPY1 were correct, AtAPY1 should exhibit the substrate specificity typical of Golgi apyrases. Therefore, the activity of AtAPY1-GFP was tested in the presence of known apyrase substrates at pH 6.5 which equals the pH found in the Golgi. AMP is  sativa) and sycalatanus).In order to investigate the possibility that the substrate specificity was pH-dependent and that it would change in favor of ATP and ADP once AtAPY1 reached the apoplast, the activity assay of AtAPY1-GFP was repeated at pH 5.5, the pH typically found in the cell wall. HoweverOne objective was to determine if AtAPY1 was a soluble protein in the lumen of the Golgi or, as implied by the TMHMM prediction program, a GolgiAtAPY1-GFP seedlings and their purity was verified with antibodies against marker proteins for cytosolic and insoluble proteins  and denaturing (4 M urea) conditions. Peripheral proteins are removed from membranes by urea which disturbs protein-protein interactions or by high salt and alkaline treatments which disrupt electrostatic and hydrophobic interactions, respectively. Except for trace amounts, the salt and Na2CO3 treatment did shift AtAPY1 from the pellet fraction to the supernatant  1.0 suggesten medium. This exn medium. This ren medium. The simn medium.in vitro germinated pollen also showed that the protein exhibiting apyrase activity in the pollen germination medium was soluble because it was measureable in siphoned off aliquots[The experiment with the aliquots. Since AAtAPY1 and AtAPY2 have been implicated to be the regulators of eATP signals in Arabidopsis,15,77-79. [AtAPY2 that could result in different localizations of the corresponding proteins. The fact that transgenic Arabidopsis plants overexpressing AtAPY2 show less eATP-mediated superoxide production than WT[Even though AtAPY1 did not agree with the profile of an ecto-apyrase, AtAPY2 remains a candidate. However, Dunkley et al.  noted a . . Neverth than WT points tSince AtAPY1 was not detected in the cell wall by three subcellular detection methods and did not exhibit the substrate specificity of an ecto-apyrase, we hypothesize that some apyrase other than AtAPY1 is the regulatory enzyme observed in eATP signaling of Arabidopsis.Pisum sativum[i by an integral membrane protein facing the Golgi lumen with its active site. It was also shown in P. sativum that UDP inhibits glycosyltransferases[A diphosphatase activity was described in the Golgi of m sativum. UDP wassferases, which fsferases in a feesferases. A role sferases, no rootsferases could beSaccharomyces cerevisiae mutant Δgda1. This yeast mutant lacks the Golgi apyrase guanosine diphosphatase 1 (GDA1) leading to a glycosylation defect[AtAPY1[xMore evidence for a function in glycosylation comes from a complementation experiment with the yeast n defect which ist[AtAPY1.It is also believed that Golgi apyrases provide the nucleoside monophosphates which serve as the substrates in exchange for nucleotide sugars from the cytosol in an antiport mechanism. TherefoAtAPY1-SNAP or -GFP allowed specific labeling of AtAPY1 at the subcellular level. The cell organelles exhibiting the AtAPY1-specific fluorescent signals were identified as the Golgi apparatus by the following criteria: (i) morphology, (ii) size, (iii) lack of co-localization with the endocytic marker stain FM4-64 and trans-Golgi marker protein Rab E1d, but (iv) co-localization with the three Golgi marker proteins MEMB12, SYP32 and Got1p homolog. In addition, Golgi stacks were immunolabeled with α-GFP antibodies. While this paper was under review, the Golgi localization of AtAPY1 was independently confirmed by proteome analysis of Golgi membranes and by co-localization of AtAPY1-YFP with the CFP-labeled cis-Golgi marker α-mannosidase I in transiently transformed onion peels[The use of two different transgenic lines expressing either on peels.Although it can never be ruled out that the missing detection of extracellular AtAPY1 is a matter of methodological sensitivity, our results show that AtAPY1 is primarily present in the Golgi. Therefore, AtAPY1 is highly unlikely to represent the regulatory enzyme of eATP levels,15,77,78i: Inorganic phosphate; PM: Plasma membrane; PPT: Phosphinothricin; RFP: Red fluorescent protein; RT: Room temperature; S: Supernatant; SNAP: O6-alkylguanine-DNA alkyltransferase; SPIK: Shaker pollen inward K+ channel; SYP32: Syntaxin of plants 32; SKO: Single knockout; T-DNA: Transfer DNA; TBS: Tris buffered saline; TEM: Transmission electron microscopy; TM: Transmembrane domain; TSA: Tyramide signal amplification; UA: Uranyl acetate; UDP: Uridine diphosphate; v: Version; WT: Wild type or wild-type; YFP: Yellow fluorescent protein.aa: Amino acid(s); ACR: Apyrase conserved region; AEBSF: 4-(2-aminoethyl)-benzensulfonyl fluoride hydrochloride; apyrase: Adenosine pyrophosphatase; CLSM: Confocal laser scanning microscopy; DKO: Double knockout; eATP: Extracellular ATP; ECM: Extracellular matrix; FITC: Fluorescein isothiocyanate; FM4-64: N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)-pyridinium dibromide; GDP: Guanosine diphosphate; GFP: Green fluorescennt protein; Got1p: Golgi transport 1 protein; HeNe: Helium-neon; HRP: Horseradish peroxidase; IDP: Inosine diphosphate; MC: Methyl cellulose; MEMB12: Membrin 12; MS: Murashige and Skoog; MVB: Multivesicular body; NTPDase: Nucleoside triphosphate diphosphohydrolase; ORF: Open reading frame; P: Pellet; PB: Phosphate buffer; PBS: Phosphate buffered saline; PFA: Paraformaldehyde; PAtAPY1-GFP expressing seedlings. IS conceived of the study, generated the AtAPY1-SNAP transgenic lines and the lines co-expressing AtAPY1-GFP and a marker gene, performed the genetic complementation of the DKO-GFP mutants and drafted the manuscript. All authors read and approved the final manuscript.MS carried out the molecular genetic studies of the DKO-SNAP mutants, performed the localization studies including confocal laser scanning microscopy and image analysis, sample preparations and treatments, analyzed the solubility of the AtAPY1-GFP and participated in the drafting of the manuscript. CM performed the substrate specificity analyses of AtAPY1-GFP. TK conducted the localization studies by immunogold labeling and by indirect immunofluorescence with AtAPY1-SNAP DNA sequence. The AtAPY1-SNAP DNA sequence present in the AtAPY1::AtAPY1-SNAP transgenic lines is shown. Click here for fileAtAPY1-GFP DNA sequence. The AtAPY1-GFP DNA sequence present in the 35S::AtAPY1-GFP transgenic lines is shown. Click here for fileImmunofluorescence of pre-imbedding labeled AtAPY1-GFP in root hair. Roots from AtAPY1-GFP expressing seedlings were immunostained whole mount, embedded in resin and sectioned. The sections were successively incubated with α-GFP and secondary α-rabbit Fab fragments coupled with Alexa Fluor 488. A 3-μm cross-section of a root hair is shown. The Alexa Fluor 488 fluorescence is shown in green. Scale bar equals 20 μm. Click here for fileLive imaging of AtAPY1-GFP in various cell types. CLSM images of various tissues in AtAPY1-GFP expressing seedlings show the GFP signals in green overlaid with bright field view. (A) Cotyledon epidermis, (B) hypocotyl and (C) root tip. Scale bars = 20 μm. Click here for fileSpecificity of the imaging settings for the detection of GFP and YFP fluorescence. The epidermis of cotyledons from transgenic plant lines was imaged with the GFP and YFP settings outlined under Methods for the “CLSM”. The GFP and YFP fluorescence is shown in green and magenta, respectively. Scale bars = 20 μm. (A) The identical epidermal section with two guard cells from plants expressing AtAPY1-GFP only was imaged sequentially with the GFP and YFP settings. Dot-like signals appeared in the GFP detection channel only. Only weak autofluorescence of the thickened cell wall around the stomate was visible in the YFP detection channel. (B) The identical epidermal section with two guard cells from plants expressing YFP-SYP32 only was imaged sequentially with the GFP and YFP settings. Here, only very weak signals were detectable in the GFP detection channel, but strong YFP fluorescence appeared with the YFP-specific excitation and detection. Click here for fileCo-localization analysis of AtAPY1-GFP and YFP-Got1p homolog. CLSM images of epidermal cells of cotyledons from transgenic lines co-expressing AtAPY1-GFP and YFP-Got1 phomolog were taken. The GFP fluorescence is shown in green and the YFP fluorescence in magenta. The bright field-type image was acquired with the transmitted light detector. The fluorescence signals for AtAPY1-GFP and YFP-Got1p homolog were detected separately and merged for co-localization with the “Co-localization Finder” plugin of ImageJ. Co-localization of the two proteins is depicted as white signals. The corresponding scatterplot was analyzed with the ImageJ “Colocalization Threshold” and “Coloc2” tool from ImageJ. The x-axis represents the pixel intensities from the GFP channel and the y-axis from the YFP channel. Rr = Pearson’s correlation coefficient. Scale bar = 20 μm. Click here for fileSubstrate specificity of AtAPY1-GFP at pH 5.5. The activity of AtAPY1-GFP at pH 5.5 in the presence of various substrates was measured. No activity was detectable with AMP as substrate (data not shown). Different letters above the columns indicate mean values that are significantly different from one other . Error bars represent standard deviations of two phosphate measurements from one reaction (see Methods). Abbreviation: Pi, inorganic phosphate. Click here for fileAnalysis of the purity of microsomal membrane and soluble protein fractions. Protein extracts from transgenic plants expressing 35S::AtAPY1-GFP were treated with either extraction buffer, 0.2% SDS, 2 M NaCl, 0.2 M Na2CO3 or 4 M urea and then centrifuged at 100,000 g to obtain microsomal membrane fractions (P100) and supernatants (S100). Proteins from each fraction (40 μg each) were subjected to Western blot analysis. The enrichment of microsomal and insoluble proteins in the P100 fractions and of soluble proteins in the S100 fractions was confirmed with antibodies against marker proteins. The 37-kDa cytosolic fructose-1,6-bisphosphatase (cFBPase) served as a marker protein for soluble proteins. Actin (45 kDa) was used as a marker protein for insoluble proteins under actin polymerizing conditions found in the extraction buffer and 2 M NaCl[2CO3, 0.2% SDS and 4 M urea[2 M NaCl and as a4 M urea,90. Click here for file