Subsecond Changes Research Articles

Dynamic patterns of functional connectivity in the human brain underlie individual memory formation

Remembering our everyday experiences involves dynamically coordinating information distributed across different brain regions. Investigating how momentary fluctuations in connectivity in the brain are relevant for episodic memory formation, however, has been challenging. Here we leverage the high temporal precision of intracranial EEG to examine sub-second changes in functional connectivity in the human brain as 20 participants perform a paired associates verbal memory task. We first identify potential functional connections by selecting electrode pairs across the neocortex that exhibit strong correlations with a consistent time delay across random recording segments. We then find that successful memory formation during the task involves dynamic sub-second changes in functional connectivity that are specific to each word pair. These patterns of dynamic changes are reinstated when participants successfully retrieve the word pairs from memory. Therefore, our data provide direct evidence that specific patterns of dynamic changes in human brain connectivity are associated with successful memory formation.

Neural Activity in the Anterior Insula at Drinking Onset and Licking Relates to Compulsion-Like Alcohol Consumption.

Much remains unknown about the etiology of compulsion-like alcohol drinking, where consumption persists despite adverse consequences. The role of the anterior insula (AIC) in emotion, motivation, and interoception makes this brain region a likely candidate to drive challenge-resistant behavior, including compulsive drinking. Indeed, subcortical projections from the AIC promote compulsion-like intake in rats and are recruited in heavy-drinking humans during compulsion for alcohol, highlighting the importance of and need for more information about AIC activity patterns that support aversion-resistant responding. Single-unit activity was recorded in the AIC from 15 male rats during alcohol-only and compulsion-like consumption. We found three sustained firing phenotypes, sustained-increase, sustained-decrease, and drinking-onset cells, as well as several firing patterns synchronized with licking. While many AIC neurons had session-long activity changes, only neurons with firing increases at drinking onset had greater activity under compulsion-like conditions. Further, only cells with persistent firing increases maintained activity during pauses in licking, suggesting roles in maintaining drive for alcohol during breaks. AIC firing was not elevated during saccharin drinking, similar to lack of effect of AIC inhibition on sweet fluid intake in many studies. In addition, we observed subsecond changes in AIC neural activity tightly entrained to licking. One lick-synched firing pattern (determined for all licks in a session) predicted compulsion-like drinking, while a separate lick-associated pattern correlated with greater consumption across alcohol intake conditions. Collectively, these data provide a more integrated model for the role of AIC firing in compulsion-like drinking, with important relevance for how the AIC promotes sustained motivated responding more generally.

Dynamic expectations: Behavioral and electrophysiological evidence of sub-second updates in reward predictions

Expectations are often dynamic: sports fans know that expectations are rapidly updated as games unfold. Yet expectations have traditionally been studied as static. Here we present behavioral and electrophysiological evidence of sub-second changes in expectations using slot machines as a case study. In Study 1, we demonstrate that EEG signal before the slot machine stops varies based on proximity to winning. Study 2 introduces a behavioral paradigm to measure dynamic expectations via betting, and shows that expectation trajectories vary as a function of winning proximity. Notably, these expectation trajectories parallel Study 1’s EEG activity. Studies 3 (EEG) and 4 (behavioral) replicate these findings in the loss domain. These four studies provide compelling evidence that dynamic sub-second updates in expectations can be behaviorally and electrophysiologically measured. Our research opens promising avenues for understanding the dynamic nature of reward expectations and their impact on cognitive processes.

Carbon Fiber Microelectrode pH Sensors with Voltammetry and Field Effect Transistors

Graphitic carbon fiber microelectrodes (CFMEs) have been used as biosensors for the detection of biomolecules such as neurotransmitters using fast scan cyclic voltammetry (FSCV). The electrodes are relatively small (7 microns in diameter), biocompatible, have high spatiotemporal resolution, and do not generally illicit an immune response. Traditionally, CFMEs have been used to detect neurotransmitters such as dopamine, serotonin, norepinephrine, and others. The shape and position of the cyclic voltammogram (CV) is a chemical fingerprint for neurotransmitter detection with the peak oxidative current being proportional to concentration. The method is ideal for measuring fast, subsecond changes of neurotransmitters such as the phasic firing of dopaminergic neurons. Recently, the method has been expanded to measure other molecules such as metals, amino acids, peptides (including neuropeptides), hormones, proteins, neuromodulators, and other biomolecules.In this study, we demonstrate the use of CFMEs as both FSCV sensors and field-effect transistor (FET) transducers for dynamic pH measurements. The electrochemical oxidation and reduction of functional groups on the surface of CFMEs affect their response over a physiologically relevant pH range. When measured with FET transducers, the sensitivity of the measurements over the measured pH range was found to be (101 ± 18) mV, which exceeded the Nernst value at room temperature of 59 mV by approximately 70 %. Finally, we validated the functionality of CFMEs as pH sensors with FSCV ex vivo in rat brain coronal slices with exogenously applied solutions of varying pH indicating that potential in vivo study is feasible. The sensitive, fast, biocompatible, and selective detection of pH fluctuations provides for a multitude of potential future applications such as the optimization of biomolecule measurement and measurement of pH in specific extracellular tumor microenvironments for cancer studies in addition to many others.

Observational Study of Clinician Attentional Reserves (OSCAR): Acuity-Based Rounds Help Preserve Clinicians' Attention.

Team rounding in the ICU can tax clinicians' finite attentional resources. We hypothesized that a novel approach to rounding, where patients are seen in a decreasing order of acuity, would decrease attentional attrition. Prospective interventional internal-control cohort study in which stop signal task testing was used as a proxy for attentional reserves. Stop signal task is a measure of cognitive control and response inhibition in addition to performance monitoring, all reflective of executive control abilities, and our surrogate for attentional reserves. The ICUs of Vanderbilt University Medical Center (site 1) and the University of Pennsylvania (site 2) from November 2014 to August 2017. Thirty-three clinicians at site 1, and 24 clinicians at site 2. Acuity-based rounding, in which clinicians round from highest to lowest acuity as determined by Sequential Organ Failure Assessment score or an equivalent acuity score. The stop signal task results of ICU staff at two sites were compared for conventional (in room order) versus novel (in decreasing order of acuity) rounding order. At site 1, the difference in stop signal reaction time change between two rounding types was -39.0 ms (95% CI, -50.6 to -27.4 ms; p < 0.001), and at site 2, the performance stop signal reaction time was -15.6 ms (95% CI, -29.1 to -2.1 ms; p = 0.023). These sub-second changes, while small, are significant in the neuroscience domain. Rounding in decreasing order of patient acuity mitigated attrition in attentional reserves when compared with the traditional rounding method.

A microfluidic electrochemical flow cell capable of rapid on-chip dilution for fast-scan cyclic voltammetry electrode calibration.

Here, we developed a microfluidic electrochemical flow cell for fast-scan cyclic voltammetry which is capable of rapid on-chip dilution for efficient and cost-effective electrode calibration. Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes is a robust electroanalytical technique used to measure subsecond changes in neurotransmitter concentration over time. Traditional methods of electrode calibration for FSCV require several milliliters of a standard. Additionally, generating calibration curves can be time-consuming because separate solutions must be prepared for each concentration. Microfluidic electrochemical flow cells have been developed in the past; however, they often require incorporating the electrode in the device, making it difficult to remove for testing in biological tissues. Likewise, current microfluidic electrochemical flow cells are not capable of rapid on-chip dilution to eliminate the requirement of making multiple solutions. We designed a T-channel device, with microchannel dimensions of 100μm × 50μm, that delivered a standard to a 2-mm-diameter open electrode sampling well. A waste channel with the same dimensions was designed perpendicular to the well to flush and remove the standard. The dimensions of the T-microchannels and flow rates were chosen to facilitate complete mixing in the delivery channel prior to reaching the electrode. The degree of mixing was computationally modeled using COMSOL and was quantitatively assessed in the device using both colored dyes and electrochemical detection. On-chip electrode calibration for dopamine with FSCV was not significantly different than the traditional calibration method demonstrating its utility for FSCV calibration. Overall, this device improves the efficiency and ease of electrode calibration. Graphical abstract.

Real-Time Cerebral Hemodynamic Response to Tactile Somatosensory Stimulation.

Recent studies in rodents suggest that somatosensory stimulation could provide neuroprotection during ischemic stroke by inducing plasticity in the cortex-vasculature relationship. While functional magnetic resonance imaging (fMRI) has shown that somatosensory stimulation increases cerebral blood flow (CBF) over several seconds, subsecond changes in CBF in the basal cerebral arteries have rarely been studied due to temporal resolution limitations. This study characterized hemodynamic changes in the middle cerebral arteries (MCAs) during somatosensory stimulation with high temporal resolution (100 samples/s) using functional transcranial Doppler ultrasound (fTCD). Pneumotactile somatosensory stimulation, consisting of punctate pressure pulses traversing the glabrous skin of the hand at 25 cm/s, was used to induce CBF velocity (CBFV) response curves. Changes in CBFV were measured in the bilateral MCAs using fTCD. All 12 subjects underwent three consecutive trials consisting of 20 seconds of stimulation followed by 5 minutes of rest. Sharp, bilateral increases in CBFV of about 20% (left MCA = 20.5%, right MCA = 18.8%) and sharp decreases in pulsatility index of about 8% were observed during stimulation. Left lateralization of up to 3.9% was also observed. The magnitude of the initial increase in CBFV showed significant adaptation between subsequent trials. Pneumotactile somatosensory stimulation is a potent stimulus that can evoke large, rapid hemodynamic changes, with adaptation between successive stimulus applications. Due to its high temporal resolution, fTCD is useful for identifying quickly evolving hemodynamic responses, and for correlating changes in hemodynamic parameters such as pulsatility index (PI) and CBFV.

Review—In Vivo Electrochemical Measurements of Norepinephrine in the Brain: Current Status and Remaining Challenges

Norepinephrine (NE), one of the major catecholamines in the brain, is involved in many physiological and behavioral processes such as stress and reward. Despite its important roles, NE remains largely unexplored compared to the other major central catecholamine, dopamine (DA). This is due in part to the diffuse distribution of NE projections throughout the brain and accessibility of NE neurons, complicating detection of the relatively low physiological NE concentrations. Recent studies have demonstrated that in vivo fast-scan cyclic voltammetry coupled with carbon-fiber microelectrodes can detect real time, subsecond changes of NE in the brains of both anesthetized and awake-behaving rats, offering a local view of NE regulation (release and clearance). Furthermore, these studies have revealed different regulatory mechanisms between NE and DA, and that these two catecholamines have functional differences as well. For the last decade, these fundamental studies provided new insights into the understudied roles of NE in reward/aversion processes, drug addiction, and related behavioral responses. However, there are still limitations in the electroanalytical determination of NE in vivo in terms of selectivity. Here, the current status of electrochemical measurements of NE and associated findings are highlighted, and remaining challenges are discussed.

Detecting sub-second changes in brain activation patterns during interictal epileptic spike using simultaneous EEG-fMRI

ObjectiveEpileptic spikes are associated with rapidly changing brain activation involving the epileptic foci and other brain regions in the “epileptic network”. We aim to resolve these activation changes using simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) recordings. MethodsSimultaneous EEG-fMRI recordings from 9 patients with epilepsy were used in the analysis. Our method employed the whole scalp EEG data to generate regressors for the analysis of fMRI data using the general linear model. ResultsWe were able to resolve, with milliseconds temporal resolution, changes in activation patterns involving suspected epileptic foci and other brain regions in the epileptic network during spike and slow wave. Using summary maps (called SSWAS maps) which show the activation frequency of voxels, we found that suspected epileptic foci tend to be significantly active during this interval. SSWAS maps also enabled the detection of the epileptic foci in 4 of 5 patients where the conventional event-timing-based analysis failed to identify. ConclusionThese findings demonstrated the efficacy of the method and the potential application of SSWAS maps to identify epileptic foci. SignificanceThe method could help resolve activation changes during epileptic spike and could provide insights into the underlying pathophysiology of these changes.

(Invited) In Vivo Electroanalytical Measurements of Norepinephrine in the Brain: Current Status and Remaining Challenges

Norepinephrine (NE), one of the major catecholamines in the brain, is involved in many physiological processes and behaviors ranging from fear and anxiety to reward. Despite its important roles, NE has largely been unexplored compared to the other central catecholamine, dopamine (DA). This is due in part to the diffuse distribution of NE projections throughout the brain and challenges of its low physiological concentration and anatomy, complicating its detection. Specifically, NE is ordinarily one to two orders of magnitude lower in concentration than that of DA and many rodent brain regions that have appreciable amounts of NE are only a few hundred microns across. Recent studies have demonstrated that fast-scan cyclic voltammetry (FSCV) coupled with carbon-fiber microelectrodes enables the detection of subsecond changes of NE in the brain of anesthetized and awake behaving rats in real time, offering a local view of NE regulation (release and clearance). Although the regulatory mechanisms of NE and DA neurotransmission have many similarities, these studies have revealed that NE has important functional differences compared to DA. These results have provided new insights into the understudied role of NE in reward/aversion processes and drug abuse, and their related behavioral responses for the last decade. However, there are still limitations in the electroanalytical determination of NE in vivo in terms of selectivity and sensitivity. Here, the current state of electrochemical measurements of NE and associated findings are highlighted and the remaining challenges are discussed.

Characterization of Fast-Scan Cyclic Voltammetric Electrodes Using Paraffin as an Effective Sealant with In Vitro and In Vivo Applications.

Fast-scan cyclic voltammetry (FSCV) is a powerful technique for measuring sub-second changes in neurotransmitter levels. A great time-limiting factor in the use of FSCV is the production of high-quality recording electrodes; common recording electrodes consist of cylindrical carbon fiber encased in borosilicate glass. When the borosilicate is heated and pulled, the molten glass ideally forms a tight seal around the carbon fiber cylinder. It is often difficult, however, to guarantee a perfect seal between the glass and carbon. Indeed, much of the time spent creating electrodes is in an effort to find a good seal. Even though epoxy resins can be useful in this regard, they are irreversible (seals are permanent), wasteful (epoxy cannot be reused once hardener is added), hazardous (hardeners are often caustic), and require curing. Herein we characterize paraffin as an electrode sealant for FSCV microelectrodes. Paraffin boasts the advantages of near-immediate curing times, simplicity in use, long shelf-life and stable waterproof seals capable of withstanding extended cycling. Borosilicate electrode tips were left intact or broken and dipped in paraffin embedding wax. Excess wax was removed from the carbon surface with xyelenes or by repeated cycling at an extended waveform (-0.4 to 1.4V, 400 V/s, 60 Hz). Then, the waveform was switched to a standard waveform (-0.4 to 1.3V, 400 V/s, 10 Hz) and cycled until stable. Wax-sealing does not inhibit electrode sensitivity, as electrodes detected linear changes in dopamine before and after wax (then xylenes) exposure. Paraffin seals are intact after 11 days of implantation in the mouse, and still capable of measuring transient changes in in vivo dopamine. From this it is clear that paraffin wax is an effective sealant for FSCV electrodes that provides a convenient substitute to epoxy sealants.

Functional electrical impedance tomography by evoked response (fEITER): Sub-second changes in brain function during induction of anaesthesia with propofol

Pollard, B. J.; Pomfrett, C. J.; Bryan, A.; Quraishi, T.; Davidson, J. L.; McCann, H. Author Information

In vivo voltammetric monitoring of catecholamine release in subterritories of the nucleus accumbens shell

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes has been used to demonstrate that sub-second changes in catecholamine concentration occur within the nucleus accumbens (NAc) shell during motivated behaviors, and these fluctuations have been attributed to rapid dopamine signaling. However, FSCV cannot distinguish between dopamine and norepinephrine, and caudal regions of the NAc shell receive noradrenergic projections. Therefore, in the present study, we examined the degree to which norepinephrine contributes to catecholamine release within rostral and caudal portion of NAc shell. Analysis of tissue content revealed that dopamine was the major catecholamine detectable in the rostral NAc shell, whereas both dopamine and norepinephrine were found in the caudal subregion. To examine releasable catecholamines, electrical stimulation was used to evoke release in anesthetized rats with either stimulation of the medial forebrain bundle, a pathway containing both dopaminergic and noradrenergic projections to the NAc, or the ventral tegmental area/substantia nigra, the origin of dopaminergic projections. The catecholamines were distinguished by their responses to different pharmacological agents. The dopamine autoreceptor blocker, raclopride, as well as the monoamine and dopamine transporter blockers, cocaine and GBR 12909, increased evoked catecholamine overflow in both the rostral and caudal NAc shell. The norepinephrine autoreceptor blocker, yohimbine, and the norepinephrine transporter blocker, desipramine, increased catecholamine overflow in the caudal NAc shell without significant alteration of evoked responses in the rostral NAc shell. Thus, the neurochemical and pharmacological results show that norepinephrine signaling is restricted to caudal portions of the NAc shell. Following raclopride and cocaine or raclopride and GBR 12909, robust catecholamine transients were observed within the rostral shell but these were far less apparent in the caudal NAc shell, and they did not occur following yohimbine and desipramine. Taken together, the data demonstrate that catecholamine signals in the rostral NAc shell detected by FSCV are due to change in dopamine transmission.

Subsecond Changes in Top–Down Control Exerted by Human Medial Frontal Cortex during Conflict and Action Selection: A Combined Transcranial Magnetic Stimulation–Electroencephalography Study

Action selection requires choosing one of all the possible conflicting action plans that are available. There is currently a debate as to whether the dorsal medial frontal cortex (dMFC) merely detects or actively resolves response conflict. We used combined on-line transcranial magnetic stimulation and electroencephalographic recording (TMS-EEG) to test whether human dMFC plays a critical causal role in conflict resolution, and whether the mechanism for such a function is via interactions with primary motor cortex. In an Eriksen flanker task, subjects discriminated the direction of the centermost arrow in an array of five, responding with the left or right hand. The lateralized readiness potential (LRP), a measure of relative levels of activity in left and right motor cortices, was also recorded. Reaction times and error rates were higher on incongruent than congruent trials, and incongruent trials produced a positive LRP deflection reflecting initial partial activation of the incorrect response. On one-half of trials, repetitive TMS was applied to left dMFC starting 100 ms before visual stimulus onset and ending 100 ms afterward. TMS disrupted performance by selectively increasing error rates on contralateral (right hand) incongruent trials. TMS also only modulated the LRP on incongruent trials, causing an increased positive deflection (associated with preparation of the incorrect response) starting 180 ms after visual stimulus onset. TMS of a control site did not interfere with behavior or motor cortical activity. dMFC has a direct causal role in resolving conflict during action selection, and the mechanism involves the top-down modulation of primary motor cortical activity.

Properties of Dopamine Release and Uptake in the Songbird Basal Ganglia

Vocal learning in songbirds requires a basal ganglia circuit termed the anterior forebrain pathway (AFP). The AFP is not required for song production, and its role in song learning is not well understood. Like the mammalian striatum, the striatal component of the AFP, Area X, receives dense dopaminergic innervation from the midbrain. Since dopamine (DA) clearly plays a crucial role in basal ganglia-mediated motor control and learning in mammals, it seems likely that DA signaling contributes importantly to the functions of Area X as well. In this study, we used voltammetric methods to detect subsecond changes in extracellular DA concentration to gain better understanding of the properties and regulation of DA release and uptake in Area X. We electrically stimulated Ca(2+)- and action potential-dependent release of an electroactive substance in Area X brain slices and identified the substance as DA by the voltammetric waveform, electrode selectivity, and neurochemical and pharmacological evidence. As in the mammalian striatum, DA release in Area X is depressed by autoinhibition, and the lifetime of extracellular DA is strongly constrained by monoamine transporters. These results add to the known physiological similarities of the mammalian and songbird striatum and support further use of voltammetry in songbirds to investigate the role of basal ganglia DA in motor learning.

Subsecond changes of global brain state in illusory multistable motion perception.

This study explored transient changes in EEG microstates and spatial Omega complexity associated with changes in multistable perception. 21-channel EEG was recorded from 13 healthy subjects viewing an alternating dot pattern that induced illusory motion with ambiguous direction. Baseline epochs with stable motion direction were compared to epochs immediately preceding stimuli that were perceived with changed motion direction ('reference stimuli'). About 750 ms before reference stimuli, Omega complexity decreased as compared to baseline, and two of four classes of EEG microstates changed their probability of occurrence. About 300 ms before reference stimuli, Omega complexity increased and the previous deviations of EEG microstates were reversed. Given earlier results on Omega complexity and microstates, these sub-second EEG changes might parallel longer-lasting fluctuations in vigilance. Assumedly, the discontinuities of illusory motion thus occur during sub-second dips in arousal, and the following reconstruction of the illusion coincides with a state of relative over-arousal.

Sub-second changes in accumbal dopamine during sexual behavior in male rats.

Transient (200--900 ms), high concentrations (200--500 nM) of dopamine, measured using fast-scan cyclic voltammetry, occurred in the nucleus accumbens core of male rats at the presentation of a receptive female. Additional dopamine signals were observed during subsequent approach behavior. Background-subtracted cyclic voltammograms of the naturally-evoked signals matched those of electrically-evoked dopamine measured at the same recording sites. Administration of nomifensine amplified natural and evoked dopamine release, and increased the frequency of detectable signals. While gradual changes in dopamine concentration during sexual behavior have been well established, these findings dramatically improve the time resolution. The observed dopamine transients, probably resulting from neuronal burst firing, represent the first direct correlation of dopamine with sexual behavior on a sub-second time scale.

High resolution evoked potential imaging of the cortical dynamics of human working memory

High resolution evoked potentials (EPs), sampled from 115 channels and spatially sharpened with the finite element deblurring method, were recorded from 8 subjects during working memory (WM) and control tasks. The tasks required matching each stimulus with a preceding stimulus on either verbal or spatial attributes. All stimuli elicited a central P200 potential that was larger in the spatial tasks than in the verbal tasks, and larger in the WM tasks than in the control tasks. Frequent, non-matching stimuli elicited a frontal, positive peak at 305 msec that was larger in the spatial WM task relative to the other tasks. Irrespective of whether subjects attended to verbal or spatial stimulus attributes, non-matching stimuli in the WM tasks also elicited an enhanced P450 potential over the left frontal cortex, followed by a sustained potential over the superior parietal cortex. A posterior P390 potential elicited by infrequent, matching stimuli was smaller in amplitude for both spatial and verbal WM tasks compared to control tasks, as was a central prestimulus CNV. These results indicate that WM is a function of a distributed system with both task-specific and task-independent components. Lesion studies and coarse temporal resolution functional imaging methods, such as PET and fMRI, tend to paint a fairly static picture of the cortical regions which participate in the performance of WM tasks. In contrast, the fine-grain time resolution provided by imaging brain function with EP methods provides a dynamic, picture of subsecond changes in the spatial distribution of WM effects over the course of individual trials, as well as evidence for differences in the activity elicited by matching and non-matching stimuli within sequences of trials. This information about the temporal dynamics of WM provides a critical complement to the fine-grain spatial resolution provided by other imaging modalities.

ADP Causes Subsecond Changes in Protein Phosphorylation of Platelets

We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched-flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.

ADP causes subsecond changes in protein phosphorylation of platelets

We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched- flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.