VISUAL AND AUDITORY Auditory Cortex Tracks Both Auditory and Visual Stimulus Dynamics Using Low-Frequency Neuronal

Phase Modulation
Abstract
Integrating information across sensory domains to construct a unified representation of multi-sensory signals is a fundamental characteristic of perception in ecological contexts. One provocative hypothesis deriving from neurophysiology suggests that there exists early and direct cross-modal phase modulation. We provide evidence, based on magnetoencephalography (MEG) recordings from participants viewing audiovisualmovies, that low-frequency neuronal information lies at the basis of the synergistic coordination of information across auditory and visual streams. In particular, the phase of the 27 Hz delta and theta band responses carries robust (in single trials) and usable information (for parsing the temporal structure) about stimulus dynamics in both sensory modalities concurrently. These experiments are the first to show in humans that a particular cortical mechanism, delta-theta phase modulation across early sensory areas, plays an important active role in continuously tracking naturalistic audio-visual streams, carrying dynamic multi-sensory information, and reflecting cross-sensory interaction in real time.

I NTRODUCTION
We do not experience the world as parallel sensory streams; rather, the information extracted from different modalities fuses to form a seamlessly unified multi-sensory percept dynamically evolving over time. There is a compelling benefit to multimodal information: behavioral studies show that combining information across sensory domains enhances unimodal detection abilityand can even induce new, integrated percepts . The relevant neuronal mechanisms have been widely investigated. One typical view posits that multisensory integration occurs at later stages of cortical processing, subsequent to unisensory analysis. This view has been supported by studies showing that higher, association areas in temporal, parietal, and frontal cortices receive inputs from multiple unimodal areas and respond to stimulation in manner that reflects multisensory convergence, for example with amplified or suppressed responses for multimodal over unimodal stimuli . A growing body of evidence provides a complementary view, suggesting that crossmodal interaction is not restricted to association areas and can occur at early, putatively

unisensory cortical processing stages . For example, non-auditory stimulation (visual and somatosensory) has been found to drive auditory cortical activity, as observed in both humans and animals . Similarly, visual cortical responses are modulated by inputs from other modalities . Importantly, independent anatomical evidence also reveals direct connections among early sensory areas. Therefore, multisensory integration may operate through lateral cross-sensory modulation, and there exist multiple integration pathways beyond purely hierarchical convergence . How is early cortical activity coordinated? Beyond the classical examination of crossmodal influences on neuronal firing rate, recent studies suggest temporal coherence to underlie multisensory integration . This view posits that oscillations synchronous across different brain areas might serve an essential role in multisensory binding, similarly as that for feature binding and attentionalselection . Several EEG/MEG studies in humans implicate oscillations and crossarea coherence in multisensory integration . However, most of the studies employed short, transient multisensory stimuli and focused on the evoked transient oscillatory power instead of examining sustained cross-modal modulation for long, naturalistic audiovisual streams. Importantly, with regard to the cross-area modulation mechanism, it has recently been suggested that cross-sensory phase modulation may underlie this interaction . For example, nonauditory inputs (re)set the phase of ongoing local neuronal activity in auditory cortex to a highexcitability state (reflected in phase angle), effectively selecting or amplifying the response to subsequent auditory inputs . Whether such a mechanism is implemented in populations of neurons and could mediate the perception of audiovisual speech in human viewers/listeners is completely unknown. In order to test directly the proposal of cross-modal phase modulation of oscillatory neural activity, we investigate online audiovisual interaction, in auditory and visual cortices simultaneously, by recording magnetoencephalography (MEG) responses from human participants presented with 30-s-long natural movie clips from the movie Dumb and Dumber (1994, New Line Platinum Series). These video segments had either matched (congruent audio-visual combinations, V1A1, V2A2, V3A3) or mixed streams (incongruent audio-visual, V1A3, V2A1, V3A2). Building on our previous results showing that the theta-band phase pattern in human auditory cortex reflects the dynamic structure of spoken sentences, we employed a new trial-by-trial phase tracking analysis to explore multi-sensory integration. We conjectured that, in response to naturalistic audio-visual streams (movies), the low-frequency phase of auditory and visual sensory activity in single trials (i) will robustly track and discriminate (in a classification analysis) the sensory stream dynamics in each modality (within-modality tracking; i.e. auditory channel tracks auditory, visual tracks visual dynamics), (ii) may carry information about stimulus dynamics in the other modality (cross-modality tracking; e.g. an auditory channel can reflect visual dynamics), and (iii) that the efficacy of such cross-sensory phase modulation (trialto-trial phase variance) depends on the relative audiovisual timing, such that a temporally

matched audio-visual stream will enhance phase tracking reliability, compared to unmatched (mixed) pairs. Our data support these predictions, highlighting the critical role of cross-sensory phase modulation of oscillations in multisensory integration, commensurate with the hypothesis . We thus argue that multi-sensory integration may use cross-modal phase modulation as a basic mechanism to construct temporally aligned representations that facilitate perceptual decoding of audiovisual speech.

R ESULTS
Low-Frequency Phase Patterns in Auditory and Visual Areas Carry Reliable Information about Audiovisual Movies We first assessed whether MEG responses in single trials can reliably track the six movie clips we presented to participants (three Matched, three Mixed). The phase and power pattern of MEG responses to the movies (see illustration of cross-trial phase coherence analysis in Figure 1a) and the corresponding discrimination ability were calculated as a function of frequency of the brain response (050 Hz) using previously developed methods. We quantified stimulus-specific trial-by-trial phase and power pattern coherence in 20 auditory and 20 visual channels, which were defined in separate auditory (1 kHz tone pip) and visual (alternating checkerboard) localizer pretests for each subject (see Figure S2). As illustrated in Figure 2a, both auditory and visual cortical responses showed good discrimination ability in the delta-theta-band (27 Hz) phase pattern (above zero discrimination score, 2-way ANOVA, main effect of frequency, F(24, 840) = 7.94, p<0.0001; post-hoc one-sample t test in delta-theta band (2~7 Hz), Auditory: t = 11.57, df = 35, p<0.0001, Visual: t = 11.16, df = 35, p<0.0001). Critically, phase tracking was not accompanied by comparable power pattern tracking (Figure 2b, 2-way ANOVA, main effect of frequency, F(24, 840) = 0.517, p = 0.97; ttest in delta-theta band (2~7 Hz), Auditory: t = 0.913, p = 0.368; Visual: t = 0.698, p = 0.49). These results demonstrate that the phase of ongoing auditory and visual cortical low-frequency oscillations is reliably modulated by the audio-visual stimuli, and thus conveys information about the rich naturalistic dynamics of these multi-sensory movies.

DISCUSSIONS
We examined multi-sensory interaction in early sensory areas in MEG responses recorded from human subjects viewing and listening to natural audio-visual movies. We show that the low-frequency, delta and theta phase pattern in early visual and auditory cortices tracks (and can discriminate among) naturalistic visual and auditory stimuli, respectively, in single MEG response trials. In addition, the low-frequency phase pattern in one sensory domain can, to some extent, represent and track the stimulus structure of the other modality. Importantly, temporally aligned audio-visual streams (matched) elicit stronger low-frequency trial-by-trial phase response reliability than non-aligned streams (mixed), supporting an active cross-modal

phase modulation versus a passive stimulus following response interpretation. Finally, the delta-theta phase clusters for stronger phase tracking, indicating that it is phase resetting to the preferred or optimal phase that tracks the within-modality and across-modality stimulus structure. Congruent multisensory stimuli lead to mutual driving towards optimal phase more reliably, perhaps to achieve temporally optimized cross-sensory enhancement. We conjecture that the ongoing phase pattern of slow oscillatory activity in sensory cortices provides a unified temporal frame of reference in which continuous multi-sensory streams are seamlessly represented and integrated into a coherent percept. SUMMARY: When faced with ecologically relevant stimuli in natural scenes, our brains need to coordinate information from multiple sensory systems in order to create accurate internal representations of the outside world. Unfortunately, we currently have little information about the neuronal mechanisms for this cross-modal processing during online sensory perception under natural conditions. Neurophysiological and human imaging studies are increasingly exploring the response properties elicited by natural scenes. In this study, we recorded magnetoencephalography (MEG) data from participants viewing audiovisual movie clips. We developed a phase coherence analysis technique that capturesin single trials of watching a moviehow the phase of cortical responses is tightly coupled to key aspects of stimulus dynamics. Remarkably, auditory cortex not only tracks auditory stimulus dynamics but also reflects dynamic aspects of the visual signal. Similarly, visual cortex mainly follows the visual properties of a stimulus, but also shows sensitivity to the auditory aspects of a scene. The critical finding is that cross-modal phase modulation appears to lie at the basis of this integrative processing. Continuous cross-modal phase modulation may permit the internal construction of behaviorally relevant stimuli. Our work therefore contributes to the understanding of how multisensory information is analyzed and represented in the human brain. REACTION: In the auditory localizer pretest, the large electrophysiological response peak with latency around 100 ms after tone-pip onset was determined (M100 or N1m) and the 20 channels with largest response amplitude were defined as the auditory channels. These channels, unsurprisingly, largely lie over the temporal lobe. In the visual localizer pretest, the 20 channels with largest response amplitude at the response peak with latency around 150 ms were selected as visual channels (typically occipital). The channel selection procedure was performed for each subject separately, and all subsequent analysis was done on those independently selected channels to represent auditory and visual cortical activity, respectively. There was no overlap among the channel groups. http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000445

INFLAMMATION AND IMMUNOLOGY Aging prolongs inflammatory marker expression in regenerating rat skeletal muscles after injury
Abstract
Some of the most serious consequences of normal ageing relate to its effectson skeletal muscle, particularly significant wasting and associated weakness, termedsarcopenia. The underlying mechanisms of sarcopenia have yet to be elucidatedcompletely but an altered muscle inflammatory response after injury is a likely contributingfactor. In this study we investigated age-related changes in the expression of numerousinflammatory markers linked to successful muscle regeneration. Methods: Right extensor digitorumlongus (EDL) muscles from young (3 month), adult (12month) and old (24 month) male F344 rats were injected with bupivacaine hydrochloride tocause complete muscle fibre degeneration, then excised 12, 24, 36, and 72 hours later (n =5/age group/time point). We used qRT-PCR to quantify the mRNA expression levels of theinflammatory markers TNF , IFN , IL1, IL18, IL6, and CD18 as well as regenerativemarkers MyoD and myogenin. Results: Inflammatory markers were all increased significantly in all age groups aftermyotoxic injury. There was a trend for expression of inflammatory markers to be higher in uninjured muscles of old rats, especially at 72 hours post injury where the expression levelsof several markers was significantly higher in old compared with young and adult rats. Therewas also a decrease in the expression of regenerative markers in old rats at 72 hours postinjury. Conclusion: Our findings identify a prolonged inflammatory signature in injured muscles from old compared with young and adult rats together with a blunted expression of keymarkers of regeneration in muscles of old rats. Importantly, our findings identify potentialtargets for future therapeutic strategies for improving the regenerative capacity of skeletalmuscle during ageing. 3

Introduction
Skeletal muscle regeneration is a complex process composed of three stages: (1)myofibre degeneration; (2) inflammation; and (3) myofibre regeneration and involves the activation of quiescent satellite cells which, through the processes of proliferation anddifferentiation, participate in the reconstitution of damaged tissues [1,2]. Successful skeletalmuscle regeneration after injury requires a carefully regulated inflammatory response to remove cell debris and initiate the activation of the normally quiescent satellite cells [3]. It iswidely accepted that inflammation is a natural response to acute skeletal muscle injury asblocking inflammatory cell function by various methods has been demonstrated to result inpoor muscle regeneration [4-6].

However, what constitutes an appropriate inflammatoryresponse versus a damaging response remains poorly understood.During the early inflammatory response to injury, the most abundant immune cell typesat the injury site are neutrophils which play two roles. Firstly, they play a phagocyticfunction to clear the injury of necrotic cells, and secondly they are thought to enhance theinflammatory response via the release of pro-inflammatory cytokines such as interleukin6(IL-6) and tumor necrosis factor-alpha (TNF ) [2]. However, there is also evidence tosuggest that neutrophils may subsequently cause secondary damage to the muscle asneutrophil depletion and/or manipulation of neutrophil function have been demonstrated toreduce muscle damage [2]. Inflammatory cell infiltration, including both neutrophils andmacrophages, is associated with an increase in a number of pro-inflammatory cytokines such asIL-1 , tumor necrosis factor (TNF), and transforming growth factor beta-1 (TGF- 1). Asecondary phase of the inflammatory response is indicated by an influx of macrophages,which appears to coincide with a decline in neutrophil numbers. Macrophages play a role in 4the removal of necrotic debris and produce soluble factors that promote regeneration [2].Previous studies in rats have revealed that 2-3 days after lengthening contraction-inducedinjury, there is a temporary increase in ED1+(infiltrating) macrophage content in damagedmuscles followed by a transient rise in ED2+(resident) macrophages, which are associated withmuscle regeneration and repair [7,8].In mammals, ageing is associated with a progressive decline in skeletal muscle massand function, called sarcopenia [9]. Muscles of old animals are more susceptible to injury,they regenerate poorly, and functional recovery remains incomplete [10-13]. Cycles ofdamage and less-than-complete repair (due to decreases in circulating anabolic hormones andgrowth factors) cause muscle atrophy and weakness with age. Furthermore, chronic lowgrade systemic inflammation and prolonged inflammatory responses to infection and(muscle) injury are thought to contribute to decreased muscle protein synthesis and reducedregenerative capacity in the elderly, given that increased amounts of circulating proinflammatory cytokines, such as IL-6 and TNF- , have been associated with muscle wasting.The impact of ageing on the inflammatory/cytokine response of human skeletal musclesafter injury remains poorly understood, but there is strong evidence indicating theimportance of inflammatory factors in the onset and progression of age-related musclewasting. Comparison of the molecular signature between young and old malesusing microarrays, revealed an increased expression of several inflammatory and apoptoticgenes [14]. Age-related differences in the expression levels of TNF- , IL-6, CD18, andTGF- 1 have also been demonstrated after an acute bout of eccentric exercise [15].However, in that study, both young and old subjects were exercised at a similar percentage ofVO2max, and so given that older subjects have a lower functional capacity, the mechanicalforces placed on the muscles may have differed significantly between the groups, which 5could have contributed to the different amounts of muscle damage and resulting cytokineresponse.

Discussion
Although a number of different hypotheses have been proposed to explain the cause of the age related impairment in skeletal muscle regeneration, the exact mechanisms remainelusive. In this study, we provide new evidence for an extended inflammatory signature ininjured muscles from old compared with young and adult rats; and a blunted expression ofkey markers of regeneration in injured muscles from old rats. These new findings addconsiderably to our understanding of age-related changes to mammalian skeletal muscle andthe regenerative responses after injury.There is considerable evidence for altered inflammatory cytokine signaling in skeletalmuscles with ageing [15,17,22,23-25]. The plasma concentration of cytokines that playcentral roles in the early stages of the pro-inflammatory response, such as IL-6 and TNF , areincreased in elderly individuals, and these are correlated significantly with an ageassociatedincrease in adipose [26] and a concomitant decrease in muscle strength [23,26]. Thisobservation was different from that of Hamada and colleagues who reported no difference in 11TNF levels between active young and old individuals before exercise, although adiposecontent was not taken into account in this study and could explain the disparity in theobservations reported [15]. The findings of the present study are consistent with an increasein basal TNF mRNA expression in uninjured contralateral EDL muscles [23,26] and supportstrongly the hypothesis that ageing is associated with an increase in cytokine levels. Other studies have shown decreased levels at rest and impaired responses to exercise for mRNAs for factors implicated in muscle growth and development [27,28]. In young and old mensubjected to resistance exercise, expression of IL-1 was not different between groups atbaseline but increased within 24 hours of the exercise bout only in young subjects. Thesefindings and those from related studies suggest that pro- and anti-inflammatory cytokineresponses to damaging exercise are impaired with ageing [28,29]. Yet despite studies suggesting a link between inflammation and ageing, the direct effect of inflammation onmuscle mass and strength has onlybeen demonstrated recently. In a model of local muscleinflammation where casein was injected directly into soleus muscles of rats, there was asignificant reduction in muscle mass and strength [30] which was only fully restored 2 weeksafter resolution of the inflammatory response. Considering the age-related increase ininflammation and reduced muscle fibre regeneration after injury, one interpretation is that thebiological switch that regulates the inflammatory response is impaired with ageing.Following the inflammatory response, activation of normally quiescent satellite cells isintegral to the processes of repair and growth of skeletal muscle [31]. These processes aremodulated by a variety of local factors, including the family of transcription factors known asmyogenic regulatory factors (MRFs) (MyoD, and myogenin) [32]. Although MyoD andmyogenin are barely detectable in skeletal muscles of adult animals [33], elevated mRNA of MyoD and myogenin has been demonstrated in skeletal muscles of aged animals [33,34],whereas protein levels are blunted in muscles from aged rats [35,36]. It should be noted, 12however, that these findings are based on basal levels or levels measured immediately after asingle bout of exercise. Only one study has investigated the MRF profile in a model ofsenescence. Bigot and

colleagues [37] showed that in the process of differentiation frommyoblast to myotube, mRNA expression of a number of MRFs were reduced significantlyand/or delayed in senescent human myoblasts. Activation of myogenin was delayed by 15hours and reduced 7-fold in senescent myoblasts compared with those from young, whereasMyoD expression was 3.3-fold lower in senescent cells. Although the results from theseexperiments support the findings of the present study, it should be noted that they were conducted in the absence of a chronic inflammatory signal, indicating that there are otherfactors besides altered inflammation that also contribute to the age-related decline in muscleregenerative capacity.It should be noted that different models of injury are likely to result in differences inmuscle inflammatory responses. In the present study, we used a myotoxic model of injurywhere the damage was limited only to muscle fibres and not to other structures. Othermodels, such as whole muscle grafting [17] are likely to elicit a different inflammatorysignature and MRF expression during regeneration given that the blood supply to the graftedmuscle is compromised and successful revascularisation is necessary for successful musclerepair.Our findings revealed that the altered inflammatory response was linked with a bluntedmuscle regenerative response in aged rats. We found that the basal levels of inflammatorycytokines are higher in muscles from old rats and the inflammatory response followingmyotoxic injury was prolonged in old compared with young and adult rats. These findingsare consistent with the notion of aberrant inflammatory cytokine signaling in skeletal muscleageing and that loss of the inflammatory regulation contributes to the age-related impairmentin muscle regenerative capacity. http://www.journal-inflammation.com/content/pdf/1476-9255-8-41.pdf

SUMMARY: Although a number of different hypotheses have been proposed to explain the cause of the age related impairment in skeletal muscle regeneration, the exact mechanisms remainelusive. In this study, we provide new evidence for an extended inflammatory signature ininjured muscles from old compared with young and adult rats; and a blunted expression ofkey markers of regeneration in injured muscles from old rats. These new findings addconsiderably to our understanding of age-related changes to mammalian skeletal muscle andthe regenerative responses after injury.There is considerable evidence for altered inflammatory cytokine signaling in skeletalmuscles with ageing. The plasma concentration of cytokines that playcentral roles in the early stages of the pro-inflammatory response, such as IL-6 and TNF , areincreased in elderly individuals, and these are correlated significantly with an age-associatedincrease in adipose and a concomitant decrease in muscle strength.

REACTION: Inflammatory markers were all increased significantly in all age groups afterMyotoxic injury. There was a trend for expression of inflammatory markers to be higher inuninjured muscles of old rats, especially at 72 hours post injury where the expression levelsof several markers was significantly higher in old compared with young and adult rats. Therewas also a decrease in the expression of regenerative markers in old rats at 72 hours postinjury.

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MA.RUZETTE C. CATUGDA BSN3B