Resting State Networks Awake Rats Figure 2. Reproducibility Analysis with RAICAR_N. The top panel displays components sorted according to reproducibility level. The red line marks the 90% cutoff as determined in the top panel. With a 90% cutoff, 7 components are above the threshold; with a 95% cutoff 6 components survive. The bottom panel depicts the normalized reproducibility and the adjusted cut-off for 90%. doi:10.1371/journal.pone.0025701.g002 statistically based on their probability of reproducibility. The networks could serve as a base to further study changes induced pharmacologically or in disease models. Methodological Approach Performing awake animal imaging eliminates issues related to anesthesia [21,36]. It might introduce other issues such as restraint-induced stress. However, this approach utilizes 3 training sessions, which has been shown to alleviate stress in the animals. King et al. have shown that stress hormone levels in the restraint rodent are close to unrestraint values after 3 trainings [37]. Our previous work in a different group of animals indicated that heart rate during scanning was within the normal range and hence taken as an indication of absence of high stress in the animal [30]. The approach utilized here allows objective identification of the most reproducible RSN components. Nevertheless, it is possible that some biologically relevant components are only moderately reproducible and, hence, this analysis might miss those compo- nents. In principle, they could be recovered by relaxing the p-value threshold on reproducibility. Furthermore, the degree of repro- ducibility does not necessarily imply higher biological importance. Awake Rat RSNs Resting state networks represent low frequency brain fluctua- tions that correspond to functionally relevant networks [8,15]. They define long distance interactions in the brain among brain substrates and are associated (in humans) with specific brain tasks at rest such as monitoring and reporting state of self, orientation, and interpretation of environment [9]. From a biological perspective, an anesthetized rat might have some of these networks’ functions compromised and their measurement under anesthesia might not reflect actual brain networks [36]. For instance, one of the networks observed in awake rodents [27] is associated with integration of sensory input, cognitive processing, and output. If the rodent is unconscious, it is difficult to assess the significance of the activity of such a network, although integration might still take place because of baseline neuronal activity [36]. In an awake animal, brain function probably requires a number of ‘‘networks’’ that would relate to the ability to interact with the environment in terms of survival similar to that in humans. These we suggest would include (1) interoception with involvement of regions such as the insula and cingulate cortices; (2) exteroception with conscious appreciation (somatosensory cortex, thalamus) of environmental sensory cues and the ability to move toward or away (motor systems) from these cues; (3) emotional processing with the ability to evaluate emotional salience of stimuli – may include regions such as the amygdala; (4) autonomic function involving areas that include the hypothalamus; (5) general awareness – default mode network; and (6) endogenous modula- tion of sensory inputs (e.g., pain). It is unlikely that a single brain rhythm is associated with specific brain functions and overlap of brain regions with different components (see [38]. In our study, seven robust and reproducible components were found in awake rats. The 7 observed networks seem to be relevant and show some similarity to the networks observed in healthy humans, specifically those defined in Smith et al. [12]. We discuss these below, noted in their order of significance as noted in the results section. PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e25701