[58]. In humans, the region most closely associated with interoception is the right anterior insula [59] as well as other cortical, such as primary and secondary somatosensory, frontal and cingulate cortices [60]. Conditions essential to survival salience are components evaluated as the interoceptive state (e.g., pain, temperature, muscular and visceral sensations, vasomotor activity, hunger, thirst). In our analysis, the insula as well as the cingulate and visual cortices were noted to be significantly involved. Interactions between the insula and cingulate have been reported in humans. Within the cingulate, two systems exist; one to the anterior and one to the posterior cingulate, that together may integrate interoception and emotional processing [61]. Here, both divisions of the rat cingulate have been observed in this network (Figure S1 and Table S7). In humans, some of the observed networks relate to the visual system with structures mostly limited to the occipital lobe [12]. In rats, we have observed limited involvement of visual related brain substrates (Component 7). Components 8–20. As indicated in the results, most of them have no biological relevance, only two seem to have some biological significance and involve the CPu and the cingulate, both structures have been identified in several other networks described above. Nevertheless, these two components might reflect sub- networks within these structures that could achieve statistical significance with a larger cohort of rats. Caveats Awake Rat Imaging. Performing studies in awake rats might induce changes in ‘‘natural’’ RSNs, as restraining could influence the animal’s stress as well as emphasize certain networks such as environmental monitoring. As described above, stress was minimized by repeated exposure of the animals to restraint and scanner noises. Excessive (chronic) restraining, however, might be inductive of constant, elevated stress [62]. At higher magnetics fields (11.7 T) it has been shown that physiological noise may impact RSNs [63]; however, it is reasonable to expect that induced physiological noise will be accounted for by an ICA analysis. Translational Equivalence. Given the obvious differences in brain morphology across species, comparisons with humans should be taken with caution. The observed networks in rodents seem to parallel those in humans. However, their assignments or interpretation should be evaluated by altering them (chemically or genetically, for example) and relating behavioral changes with RSN changes. Conclusions Robust reproducible networks in awake rats were identified. Some are in agreement with those observed in other rat RSN studies. Several of the observed networks are similar to those observed in healthy humans. The elimination of the use of anesthesia might enhance significantly the opportunity to study brain alterations in well-controlled preclinical models. This approach may provide a useful addition to the use of imaging in evaluating drug effects, disease states, or genetic and strain differences. Supporting Information Figure S1 Resting State Networks in Awake Rats. Complete maps for Components (C1–C7). All components have been thresholded according to a mixture model approach-see Methods for details. The atlas is based on the Paxinos Atlas (Paxinos and Watson). Key: Ins: Insula, AcB: Nucleus Accumbens, Motor: Motor Cortex, Amyg: Amygdala, Parab: Parabrachial, CPu: Caudate-Putamen, PAG: Periaqueductal Gray, Cereb: Cerebellum, Resting State Networks Awake Rats ParA: Parietal Association Cortex, Cnf: Cuneiform nucleus, Som: Somatosensory Cortex, Ent: Entorhinal Cortex, SupColl: Superior Colliculus, FC: Frontal Cortex, Thal: Thalamus, TpA: Temporal Association Cortex, Hypo: Hypothalamus, cing: Cingulate cortex (anterior and retrosplenial), InfColl: Inferior Colliculus. (TIF) Figure S2 Resting State Networks in Awake Rats. Complete maps for Components (C8–C13). These Components did not achieve statistical significance for reproducibility. All components have been thresholded according to a mixture model approach-see Methods for details. (TIF) Figure S3 Resting State Networks in Awake Rats. Complete maps for Components (C14–C20). These Components did not achieve statistical significance for reproducibility. All components have been thresholded according to a mixture model approach-see Methods for details. (TIF) Table S1 Table of Activations for Component 1. The Table lists the most significant activated structures for the Cerebellar Network. Structures were identified using the Paxinos Atlas [33]. Structures are listed according to the fraction of the structure being active and the statistical significance of the activation (See Methods Section). (DOCX) Table S2 Table of Activations for Component 2. The Table lists the most significant activated structures for the Visuo- Spatial System. Structures were identified using the Paxinos Atlas [33]. Structures are listed according to the fraction of the structure being active and the statistical significance of the activation (See Methods Section). (DOCX) Table S3 Table of Activations for Component 3. The Table lists the most significant activated structures for the Basal Ganglia-Hypothalamus Network. Structures were identified using the Paxinos Atlas [33]. Structures are listed according to the fraction of the structure being active and the statistical significance of the activation (See Methods Section). (DOCX) Table S4 Table of Activations for Component 4. The Table lists the most significant activated structures for the Basal Ganglia-Thalamus-Hippocampus Network. Structures were iden- tified using the Paxinos Atlas [33]. Structures are listed according to the fraction of the structure being active and the statistical significance of the activation (See Methods Section). (DOCX) Table S5 Table of Activation for Component 5. The Table lists the most significant activated structures for the Autonomic Network. Structures were identified using the Paxinos Atlas [33]. Structures are listed according to the fraction of the structure being active and the statistical significance of the activation (See Methods Section). (DOCX) Table S6 Table of Activations for Component 6. The Table lists the most significant activated structures for the Sensory Network. Structures were identified using the Paxinos Atlas [33]. Structures are listed according to the fraction of the structure being active and the statistical significance of the activation (See Methods Section). (DOCX) PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e25701