Robust Reproducible Resting State Networks in the Awake Rodent Brain Lino Becerra*, Gautam Pendse, Pei-Ching Chang, James Bishop, David Borsook Pain and Analgesia Imaging Neuroscience Group, A. A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Boston, Massachusetts, United States of America Abstract Resting state networks (RSNs) have been studied extensively with functional MRI in humans in health and disease to reflect brain function in the un-stimulated state as well as reveal how the brain is altered with disease. Rodent models of disease have been used comprehensively to understand the biology of the disease as well as in the development of new therapies. RSN reported studies in rodents, however, are few, and most studies are performed with anesthetized rodents that might alter networks and differ from their non-anesthetized state. Acquiring RSN data in the awake rodent avoids the issues of anesthesia effects on brain function. Using high field fMRI we determined RSNs in awake rats using an independent component analysis (ICA) approach, however, ICA analysis can produce a large number of components, some with biological relevance (networks). We further have applied a novel method to determine networks that are robust and reproducible among all the components found with ICA. This analysis indicates that 7 networks are robust and reproducible in the rat and their putative role is discussed. Citation: Becerra L, Pendse G, Chang P-C, Bishop J, Borsook D (2011) Robust Reproducible Resting State Networks in the Awake Rodent Brain. PLoS ONE 6(10): e25701. doi:10.1371/journal.pone.0025701 Editor: Yong He, Beijing Normal University, Beijing, China Received July 8, 2011; Accepted September 8, 2011; Published October 18, 2011 Copyright: 2011 Becerra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by National Institute of Drug Abuse (grant number K24NS064050-DB) and The Herlands Fund for Pain Research (DB, LB). The project described was supported by Award Numbers S10RR031599, 1S10RR023401, 1S10RR019307, and 1S10RR023043 from the National Center For Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Research Resources or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: becerra@nmr.mgh.harvard.edu Introduction Resting state networks (RSNs) in humans is becoming a tool to investigate brain basal states in health and disease [1–7]. Using fMRI, neuronal baseline activity of the brain is thought to measure low frequency fluctuations that are related to functionally relevant networks [8–10]. In humans these network patterns seem to be consistent and reproducible across subjects and over time [8,11– 14]. Using this approach it is possible to characterize the brain networks in terms of functionally relevant circuits that subserve specific actions such as environmental monitoring, action- execution, and sensory-perception domains [11,12,15]. Until recently, RSNs have not been studied in rodents. Consequently, healthy state basal networks are not well under- stood. Most rodent studies have been carried out on anesthetized animals. Furthermore, these studies use a seed-based approach in which specific questions are tested by using a particular brain region as a seed and determining what else in the brain displays similar temporal pattern. Kannurpatti et al. [16] used the primary somatosensory cortex (S1) as seed and found a network that consisted of bilateral S1. Zhao et al. [17] (using medetomidine anesthesia) and Majeed et al. [18] (alpha-chloralose) found S1 as well as bilateral Caudate Putamen networks (CPu) connectivity. Pawela et al., [19,20] found an extended cortical network including primary/secondary sensorimotor regions under mede- tomidine anesthesia. Two studies (Lu et al. [21] with alpha- chloralose and Wang et al. [22] with isoflorane) explored the effects of levels of anesthesia on networks and determined that connectivity decreased with increased levels of anesthesia. Similarly, in anesthetized monkeys, the networks observed in humans are also preserved [23]. These results have raised issues relating to the significance of the networks to reflect meaningful biology [24] since consciousness should be impaired under anesthesia. In a recent study by Liu et al. [25], in which fMRI (BOLD and CBF) as well as electrophysiology was used, they reported that while the level of anesthesia was light, good correlation between electrical activity and fMRI signals was observed. At deep levels of anesthesia, most burst electrical activity was suppressed but brain networks were still observed, although weaker than with light anesthesia. These findings were interpreted to reflect that at deep levels of anesthesia a loss of consciousness occurs due to a collapse of cortical activity patterns as reflected in the disappearance of electrical burst activity. The underlying coherent fluctuations that remain at deep levels of anesthesia are the ones that appear in fMRI studies, although they might not reflect brain activity. Hence, it is probable that varying types and levels of anesthetics affect spontaneous brain activity and the resulting RSNs. To the best of our knowledge, there is only one other study that explores RSNs in awake rodents [26] with a seed-based correlation method. The study used 3 seeds located at the prefrontal cortex, thalamus and retrosplenial cortex. There was a significant amount of overlap in the observed networks for the 3 seeds. The cortical ribbon encompassing sensorimotor regions was in all of them, PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e25701