There is growing interest in spontaneous fluctuations of the fMRI-BOLD signal and its potential to encode an intrinsic form of brain functional connectivity in discrete brain systems. While efforts so far have focused on clarifying the physiological basis and spatial replicability of these signals across studies 
, it is less clear how changes or differences in the behavioral state of subjects during scanning may be represented in these so-called ‘resting-state networks’ (RSNs). Such data will ultimately be important for interpreting the functional meaning of spontaneous activity fluctuations in basic and clinical fMRI studies.
The current findings add to existing work by demonstrating that measurable changes in the functional connectivity of certain RSNs emerged in healthy subjects undergoing an experimental manipulation of their subjective (i.e., mood) state during resting-like fMRI conditions 
. In each of the five common RSNs identified, we observed significant changes in their global functional connectivity pattern between the neutral and sad mood induction conditions, appearing either as regional increases or decreases of the magnitude and extent of correlated voxels in the very low frequency domain (<0.04 Hz). Changes of this nature are in good agreement with other studies to have compared fMRI steady-state and continuous task performance conditions against rest 
, but whereas in the current study, we describe these changes in the context of a multivariate analysis that has characterized changes in several networks simultaneously.
The finding of increased functional connectivity of the ‘paralimbic’ RSN during the subjective experience of sadness fits remarkably well with existing imaging studies of mood-emotion induction tasks 
, and current ideas of the functional relevance of this brain system in humans 
. This RSN consists primarily of paralimbic structures including the dorsal anterior cingulate cortex and anterior insula-operculum; regions that consistently co-activate in functional imaging studies in response to personally salient stimuli and various forms of cognitive and affective engagement 29
. The frequency and commonality of their co-activation in imaging studies has been taken to support idea that both regions represent major nodes of a cortical network for interoceptive awareness; a network thought to mediate subjective feeling states arising from brain representations of bodily responses 
. In keeping with this, the finding of increased functional connectivity of these regions in the current study during the sad memory recall may represent a general change in the subjective state of participants during this condition, rather than a specific correlate of increased sadness per se
. This would be consistent with other mood induction imaging studies, where the co-activation of these regions has also been linked to other factors, such as the degree of mental effort and cognitive demand that is evoked by different paradigms, in particular those based on emotional memory recall/recollection 
Also supporting the initial predictions of our study was the observation of reduced functional connectivity in midline regions of the ‘default mode network’ when comparing the sad recall and neutral recall conditions. This pattern appears to be consistent with the common finding of task-induced deactivations of this network that have been reported in many other functional imaging studies 
. Deactivations of this network emerge routinely in task-related imaging studies when periods of rest or passive imaging states are compared with periods of cognitively demanding tasks, implying greater functional engagement of these regions during the passive imaging states 
. This interpretation may carry over the current experiment, with the neutral recall condition representing a more passive or less cognitively demanding task than the sad memory recall, as described in other studies 
. That such a ‘cognitive’ aspect to the sad memory recall condition may have reduced functional connectivity of the default mode network is also consistent with two recent studies that have compared the functional connectivity of this network during resting-state and working memory task conditions using equivalent methods (i.e., ICA 
). However, the extent to which our findings also mark a deviation from the resting-state activity of this network was not directly tested, and thus it is possible that both task conditions may have reduced its resting functional connectivity, albeit to different extents.
In addition to the observed changes in the ‘paralimbic’ and ‘default mode’ RSNs, functional connectivity increases were also identified in association with the sad recall conditions in three other previously described RSNs, including left and right frontoparietal ‘dorsal attention’ systems and a bilateral ‘auditory cortex’ network 
. Like the aforementioned networks, there is also intuitive appeal in the nature of these findings, where for instance the greater apparent increase of functional connectivity in the left versus right frontoparietal network during sad memory recall agrees with the pattern of lateralized cortical activations that have been reported in existing imaging studies of autobiographical and emotional memory recall 
. Similarly, for the auditory cortex RSN, functional connectivity changes were mapped primarily to the mid and posterior insula cortex, which is consistent with other evidence linking this region, rather than primary auditory cortex, to the experience of emotional intensity during mental imagery 
. Nevertheless, despite the plausibility of these findings, they should also be considered preliminary at this point. This is said for two reasons: Firstly, compared to the paralimbic and default mode networks, the identification of these other RSNs demonstrated a tendency towards lower reproducibility (80%) in our simple split-half analyses shown in Figure S2
. Secondly, and importantly, we report these findings in the context of a novel and exploratory imaging analysis (group ICA) with no formal hypotheses regarding the influence of our experiment on the activity of these networks. Therefore, we suggest that these findings are in need of replication.
Extending the above discussion, we have also described variable effects of the mood induction experiment on two frequently reported sensory RSNs involving visual and sensorimotor cortices (Figure S1
). From our original group analyses (n
24), both of these networks showed lower reproducibility than other RSNs to the extent they were not detected as a reliable signal source in association with the sad memory recall condition. Although others have commented on the variability of certain RSN measurements 
, the precise interpretation of this result in our study is not straightforward. For instance, it is possible that the lower reproducibility of these two networks reflects; (i)
greater variability among subjects due to a true influence of task on their functional organization; (ii)
greater variability in the measurement of their activities because of the specific analysis approach; or (iii)
some interaction of both factors. The last two possibilities may also be considered more probable with the use of ICA algorithms compared to other functional connectivity methods (i.e. CCAs), given their greater analytic complexity (however, see 
). We explored this by performing additional CCAs and split-half analyses for both of these sensory RSNs and observed similar pattern of low reproducibility for the sensorimotor network (Figure S3
). Clearly, further and more sophisticated studies are needed to address the issue of reproducibility of different functional connectivity methods in fMRI studies and whether such approaches can be meaningfully integrated to provide a consensus imaging approach.
While evidence is accruing in support of a definite neuronal basis to the spontaneous signal fluctuations observed in fMRI experiments (for a discussion see 
), the influence of non-neuronal factors on the measurement of these signals should not be ruled out. That is, although methods such as ICA have shown good utility in separating potential noise sources in resting-state fMRI studies, including aliased physiological signals associated with the cardiac (vascular pulsation) and respiratory (chest movement) cycles 
, we cannot exclude an influence of these signals in the our study without direct physiological monitoring. We were also unable to quantify the influence of slow variations in subjects' breathing rate and volume (reflected as small variations in end-tidal arterial CO2
) on the extracted BOLD signal measurements 
. While recent work suggests that the effect of aliased physiological signals on the estimation of low frequency BOLD signal fluctuations may be small 
, it will be important for future studies to account for these effects directly with physiological monitoring.
Although much remains to be known about these slow BOLD signal fluctuations, they are becoming increasingly seen as a major source of non-modeled variance in fMRI studies that may provide some new insight into the functional connectivity or organization of discrete neuroanatomical systems 
. Our findings support the proposal of recent fMRI studies which also indicate that the functional connectivity of these resting-state networks may, in part, represent a dynamic image of the current brain state 
. Considering the growing interest of resting-state (or resting-like) fMRI protocols to compare brain functional connectivity estimates in healthy and clinical populations 
, further work investigating the state and trait behavioral correlates of these activity fluctuations is necessary.