Because both the global and RVT signals were obtained in arbitrary units, we first normalized the global and RVT signals to their mean values to derive percent changes of these signals. We then computed the energy below 0.08 Hz and the standard deviation of the percent change signals. To assess changes in the finger tapping BOLD response to caffeine, average BOLD block responses were extracted from the combined motor cortex ROI. Each subject’s BOLD block response was interpolated to a time resolution of 0.25s and the following timing parameters were computed: 1) time to reach 50% of the peak response , 2) time at which the response falls to 50% of the peak response , and 3) the full-width half-maximum . In addition, the peak BOLD response amplitudes were calculated for each subject.Figure 2.1 provides a qualitative summary of the results. Resting-state functional connectivity maps obtained by using the average signal from the left ROI as a reference are shown for a representative slice from each subject. The top two rows display each subject’s average pre-dose and post-dose maps for the caffeine session. The extent of significantly correlated voxels in the post-dose connectivity maps has been visibly diminished in most subjects, as compared to the pre-dose maps. In contrast, for most subjects the control session maps in the bottom two rows do not demonstrate an obvious difference in connectivity between pre-dose and post-dose conditions. Metrics of connectivity strength are graphed in Figure 2.2. The scatter plots in the top row show the mean z scores for each subject from the caffeine session and control session, with the solid black lines representing equality between the pre-dose and post-dose sections. The caffeine session plot shows a significant = 3.3, p = 0.01 caffeine-induced decrease in mean z score across subjects. In contrast,rolling benches the mean z scores in the control session are clustered about the centerline, without a significant = -0.51, p = 0.62 change between pre-dose and post-dose conditions.

These results are consistent with a repeated measures two-way ANOVA, which showed that the interaction between period and session is significant = 9.8, p = 0.01. In addition, the baseline mean z scores in panels and are not significantly different = 0.36, p = 0.73. The mean z scores presented here were determined using the average time course from the left motor ROI as a reference. If the average time course from the right ROI is used as a reference instead, the caffeine-induced decline in mean z score is still significant = 2.8, p = 0.02.We have shown that caffeine reduces resting-state BOLD connectivity in the motor cortex. This reduction was apparent in the connectivity maps from a representative subject and in the quantitative metrics of connectivity obtained for all subjects. In addition, we found that baseline CBF and the magnitudes of the spontaneous BOLD fluctuations were decreased by caffeine. Physiological confounds, such as changes in respiration and arterial CO2 levels, can alter BOLD signal fluctuations. Regression-based removal of global and respiration volume per time signals has been shown to reduce the influence of these physiological confounds . When we included the regression of the RVT or global signal in our data analysis we found that the post-dose connectivity metrics were still significantly decreased. In fact, RVT signal regression did not significantly alter the functional connectivity metrics . Furthermore, we found that the variance or energy in the global and RVT signals were not significantly altered by caffeine . These findings suggest that the caffeine-induced reduction in BOLD connectivity is not primarily due to respiration changes. In agreement with the literature, we found that the caffeine-induced reduction in CBF was associated with an acceleration of the BOLD response to a finger tapping task . In addition, vasoconstriction due to either hypocapnia or nitric oxide synthase blockade has been found to increase low-frequency fluctuations in CBF . We have previously presented a bio-mechanical model that explains how vasoconstriction can increase the dynamic compliance of the arterioles and thus increase the responsiveness of the vasculature to neural stimulus and fluctuations .

However, in this study we found that both the spectral amplitude of low-frequency BOLD fluctuations and the coherence between resting BOLD fluctuations were diminished by caffeine, suggesting that an increase in bio-mechanical responsiveness was not a dominant factor. As binding of adenosine to A2 receptors is associated with vasodilation, caffeine-related antagonism may reduce the ability of adenosine to contribute to functional increases in cerebral blood flow. In a recent study, we found that a 200 mg oral dose of caffeine led to a significant decrease in the absolute functional CBF change in response to a visual stimulus but resulted in a significant increase in the percent CBF change . These results indicated that the drop in the absolute functional CBF change was primarily related to a drop in baseline CBF as opposed to reflecting an impairment of neurovascular coupling. Also consistent with our prior work and the results of this study, Liau et al. did not find a significant change in the BOLD response. Taken together, these results indicate that caffeine’s effect on adenosine-related vasodilation does not significantly reduce the task-related functional BOLD response. If task-related and resting-state BOLD activity share a common neurovascular coupling pathway, then the task-related BOLD results suggest that an impairment of adenosine-related vasodilation was probably not the dominant factor in the reduced functional connectivity observed in this study. Further studies elucidating the similarities and differences in neurovascular coupling for task-related and resting-state BOLD signals would be useful. In addition to its vasoconstrictive effects, caffeine directly influences neural activity. Caffeine stimulates the central nervous system by antagonizing adenosine A1 receptors throughout the brain. This blocks the inhibitory actions of adenosine, which include hyperpolarization of membrane potentials and the inhibition of neurotransmitter release . Although caffeine acts as a neurostimulant, previous work has shown that a 200 mg dose of caffeine reduces the power of resting electroencephalography activity in the alpha, beta, and theta bands . In addition, the coherence of anterior cortex neural fluctuations in the alpha and theta bands is decreased by caffeine when compared to periods of caffeine abstinence .

Simultaneous EEG/fMRI recordings have shown that resting-state BOLD fluctuations are significantly correlated with EEG power fluctuations in the alpha band , the beta band , and the theta band . These prior findings suggest that the reduction in resting-state BOLD fluctuations and connectivity found in this study may primarily reflect changes in neural power fluctuations. Although the physiological effects of caffeine are often beneficial,rolling grow table such as enhanced mood, attention, wakefulness, and motor speed , a 200 mg dose has been shown to impair several types of memory tasks, including motor learning of a finger tapping task . In light of our findings, this observed decrease in motor learning might reflect a caffeine-induced decrease in resting state neural connectivity. Further experiments with simultaneous EEG/fMRI would be useful to determine if caffeine-induced changes in neural power fluctuations are directly related to the observed reduction of BOLD connectivity. In addition to caffeine, a number of pharmacological agents have been found to alter resting-state BOLD connectivity. Both hypercapnia and cocaine have been shown to reduce the magnitude and coherence of resting-state BOLD fluctuations, while anesthesia appears to have varying effects depending on the specific agent and brain region. Cognitive disorders such as Alzheimer’s disease, schizophrenia, multiple sclerosis, and epilepsy have also been shown to modulate BOLD connectivity . While changes in resting-state BOLD connectivity are typically interpreted as changes in coherent neural activity across spatially distinct brain regions, changes to the neurovascular system may also alter connectivity. For example, as mentioned in the Introduction, hypercapnia appears to decrease BOLD connectivity by weakening the neurovascular coupling between spontaneous neural activity and resting-state BOLD fluctuations. Since many pharmacological agents and diseases are likely to affect both the neural and vascular systems, a greater understanding of the neural and vascular mechanisms that give rise to resting-state BOLD connectivity will be critical for the correct interpretation of changes in connectivity. Similar to a prior study examining the effect of caffeine on baseline oxygen metabolism , we used a control session to examine potential changes in baseline CBF, resting-state functional connectivity, and low-frequency BOLD fluctuations that might have been caused by differences in the subject’s state between the pre-dose and post-dose scan sections. We did not find significant differences between the pre-dose and post-dose results obtained during the control session, indicating that the caffeine-induced decrease in BOLD connectivity was not due to factors, such as subject fatigue, associated with participating in two scan sections. As our protocol did not involve the administration of a placebo dose, it is possible that psychological effects associated with taking a dose could have affected the functional connectivity measures.

Future studies would be useful to assess the effect of a placebo dose on resting-state BOLD connectivity. There was a range of caffeine usage in the current sample of subjects. Prior work has demonstrated variability in the task-related BOLD response due to differences in dietary caffeine consumption. Inter-subject differences in caffeine consumption may also influence the effect of caffeine on resting-state BOLD connectivity. In addition, subjects in this study were asked to abstain from caffeine for at least 12 hours prior to being scanned. Caffeine withdrawal has been shown to alter EEG power in the alpha and theta bands . It is possible that caffeine’s effect on resting-state BOLD connectivity will differ based on the subject’s state of withdrawal during the pre-dose scan section. Further investigation of the effects of dietary consumption and withdrawal on caffeine-induced changes in BOLD connectivity will be helpful. The work presented here shows that caffeine reduces resting-state BOLD connectivity in the motor cortex, most likely by reducing the amplitude and coherence of neural power fluctuations. As the distribution of adenosine receptors varies across the brain , it is possible that the effect of caffeine on functional connectivity will vary with the local receptor concentration. While future work is necessary to determine whether caffeine alters connectivity in other functional networks, the findings of this study indicate that caffeine usage should be carefully considered in the design and interpretation of studies involving resting-state BOLD connectivity.Resting-state functional MRI can be used to assess functional connectivity within the brain through the measurement of correlations between spontaneous blood oxygenation level-dependent fluctuations in different regions. Synchronous BOLD fluctuations have been consistently found at rest within functional networks such as the motor cortex, visual cortex, and default mode network . A growing number of studies have shown that functional connectivity is altered for cognitive disorders such as multiple sclerosis, epilepsy, Parkinson’s, and Alzheimer’s disease , suggesting that resting-state studies can aid in disease diagnosis and improved understanding of disease mechanisms. In addition, inter-subject differences in functional connectivity have been shown to correlate with performance on working memory tasks and intelligence . To date, functional connectivity studies have typically employed stationary metrics obtained with seed-based correlations or independent component analysis computed over an entire resting scan. However, recent work has shown that the correlation strength between different brain regions may vary in time. For example, a study using magnetoencephalography found transient formations of widespread correlationsin resting-state power fluctuations within the DMN and task positive network . This non-stationary phenomenon was particularly apparent when considering nodes in different hemispheres, which exhibited very low stationary correlation. Another study using fMRI found that the phase angle between spontaneous BOLD fluctuations in the DMN and TPN varied considerably over time, with frequent periods of significant anti-correlation . These studies indicate that coordination of spontaneous neural activity is a dynamic process, and suggest that time varying approaches can provide critical insights into functional connectivity. Despite the increasing appearance of resting-state functional connectivity studies in the literature, it remains difficult to interpret the physiological mechanisms behind changes in BOLD signal correlations. The BOLD signal provides an indirect measure of neural activity, and is a complex function of changes in cerebral blood flow , cerebral blood volume, and oxygen metabolism . Factors that alter any part of the pathway between neural activity and the BOLD response can change functional connectivity measurements, making it difficult to decipher the origin of this effect. For example, caffeine is a widely used stimulant that has a complex effect on the coupling between neural activity and blood flow .