Natural sensory context drives diverse brain-wide activity during C. elegans mating.

Natural goal-directed behaviors often involve complex sequences of many stimulus-triggered components. Understanding how brain circuits organize such behaviors requires mapping the interactions between an animal, its environment, and its nervous system. Here, we use brain-wide neuronal imaging to study the full performance of mating by the C. elegans male. We show that as mating unfolds in a sequence of component behaviors, the brain operates similarly between instances of each component but distinctly between different components. When the full sensory and behavioral context is taken into account, unique roles emerge for each neuron. Functional correlations between neurons are not fixed but change with behavioral dynamics. From individual neurons to circuits, our study shows how diverse brain-wide dynamics emerge from the integration of sensory perception and motor actions in their natural context.

The whisking oscillator circuit.

Central oscillators are primordial neural circuits that generate and control rhythmic movements1,2. Mechanistic understanding of these circuits requires genetic identification of the oscillator neurons and their synaptic connections to enable targeted electrophysiological recording and causal manipulation during behaviours. However, such targeting remains a challenge with mammalian systems. Here we delimit the oscillator circuit that drives rhythmic whisking-a motor action that is central to foraging and active sensing in rodents3,4. We found that the whisking oscillator consists of parvalbumin-expressing inhibitory neurons located in the vibrissa intermediate reticular nucleus (vIRtPV) in the brainstem. vIRtPV neurons receive descending excitatory inputs and form recurrent inhibitory connections among themselves. Silencing vIRtPV neurons eliminated rhythmic whisking and resulted in sustained vibrissae protraction. In vivo recording of opto-tagged vIRtPV neurons in awake mice showed that these cells spike tonically when animals are at rest, and transition to rhythmic bursting at the onset of whisking, suggesting that rhythm generation is probably the result of network dynamics, as opposed to intrinsic cellular properties. Notably, ablating inhibitory synaptic inputs to vIRtPV neurons quenched their rhythmic bursting, impaired the tonic-to-bursting transition and abolished regular whisking. Thus, the whisking oscillator is an all-inhibitory network and recurrent synaptic inhibition has a key role in its rhythmogenesis.

Decoding cognition from spontaneous neural activity.

In human neuroscience, studies of cognition are rarely grounded in non-task-evoked, 'spontaneous' neural activity. Indeed, studies of spontaneous activity tend to focus predominantly on intrinsic neural patterns (for example, resting-state networks). Taking a 'representation-rich' approach bridges the gap between cognition and resting-state communities: this approach relies on decoding task-related representations from spontaneous neural activity, allowing quantification of the representational content and rich dynamics of such activity. For example, if we know the neural representation of an episodic memory, we can decode its subsequent replay during rest. We argue that such an approach advances cognitive research beyond a focus on immediate task demand and provides insight into the functional relevance of the intrinsic neural pattern (for example, the default mode network). This in turn enables a greater integration between human and animal neuroscience, facilitating experimental testing of theoretical accounts of intrinsic activity, and opening new avenues of research in psychiatry.

Coupling of activity, metabolism and behaviour across the Drosophila brain.

Coordinated activity across networks of neurons is a hallmark of both resting and active behavioural states in many species1-5. These global patterns alter energy metabolism over seconds to hours, which underpins the widespread use of oxygen consumption and glucose uptake as proxies of neural activity6,7. However, whether changes in neural activity are causally related to metabolic flux in intact circuits on the timescales associated with behaviour is unclear. Here we combine two-photon microscopy of the fly brain with sensors that enable the simultaneous measurement of neural activity and metabolic flux, across both resting and active behavioural states. We demonstrate that neural activity drives changes in metabolic flux, creating a tight coupling between these signals that can be measured across brain networks. Using local optogenetic perturbation, we demonstrate that even transient increases in neural activity result in rapid and persistent increases in cytosolic ATP, which suggests that neuronal metabolism predictively allocates resources to anticipate the energy demands of future activity. Finally, our studies reveal that the initiation of even minimal behavioural movements causes large-scale changes in the pattern of neural activity and energy metabolism, which reveals a widespread engagement of the brain. As the relationship between neural activity and energy metabolism is probably evolutionarily ancient and highly conserved, our studies provide a critical foundation for using metabolic proxies to capture changes in neural activity.

The dorsomedial prefrontal cortex prioritizes social learning during rest.

Sociality is a defining feature of the human experience: We rely on others to ensure survival and cooperate in complex social networks to thrive. Are there brain mechanisms that help ensure we quickly learn about our social world to optimally navigate it? We tested whether portions of the brain's default network engage "by default" to quickly prioritize social learning during the memory consolidation process. To test this possibility, participants underwent functional MRI (fMRI) while viewing scenes from the documentary film, Samsara. This film shows footage of real people and places from around the world. We normed the footage to select scenes that differed along the dimension of sociality, while matched on valence, arousal, interestingness, and familiarity. During fMRI, participants watched the "social" and "nonsocial" scenes, completed a rest scan, and a surprise recognition memory test. Participants showed superior social (vs. nonsocial) memory performance, and the social memory advantage was associated with neural pattern reinstatement during rest in the dorsomedial prefrontal cortex (DMPFC), a key node of the default network. Moreover, it was during early rest that DMPFC social pattern reinstatement was greatest and predicted subsequent social memory performance most strongly, consistent with the "prioritization" account. Results simultaneously update 1) theories of memory consolidation, which have not addressed how social information may be prioritized in the learning process, and 2) understanding of default network function, which remains to be fully characterized. More broadly, the results underscore the inherent human drive to understand our vastly social world.

Characteristic BOLD signals are detectable in white matter of the spinal cord at rest and after a stimulus.

We report the reliable detection of reproducible patterns of blood-oxygenation-level-dependent (BOLD) MRI signals within the white matter (WM) of the spinal cord during a task and in a resting state. Previous functional MRI studies have shown that BOLD signals are robustly detectable not only in gray matter (GM) in the brain but also in cerebral WM as well as the GM within the spinal cord, but similar signals in WM of the spinal cord have been overlooked. In this study, we detected BOLD signals in the WM of the spinal cord in squirrel monkeys and studied their relationships with the locations and functions of ascending and descending WM tracts. Tactile sensory stimulus -evoked BOLD signal changes were detected in the ascending tracts of the spinal cord using a general-linear model. Power spectral analysis confirmed that the amplitude at the fundamental frequency of the response to a periodic stimulus was significantly higher in the ascending tracts than the descending ones. Independent component analysis of resting-state signals identified coherent fluctuations from eight WM hubs which correspond closely to the known anatomical locations of the major WM tracts. Resting-state analyses showed that the WM hubs exhibited correlated signal fluctuations across spinal cord segments in reproducible patterns that correspond well with the known neurobiological functions of WM tracts in the spinal cord. Overall, these findings provide evidence of a functional organization of intraspinal WM tracts and confirm that they produce hemodynamic responses similar to GM both at baseline and under stimulus conditions.

Partial entropy decomposition reveals higher-order information structures in human brain activity.

The standard approach to modeling the human brain as a complex system is with a network, where the basic unit of interaction is a pairwise link between two brain regions. While powerful, this approach is limited by the inability to assess higher-order interactions involving three or more elements directly. In this work, we explore a method for capturing higher-order dependencies in multivariate data: the partial entropy decomposition (PED). Our approach decomposes the joint entropy of the whole system into a set of nonnegative atoms that describe the redundant, unique, and synergistic interactions that compose the system's structure. PED gives insight into the mathematics of functional connectivity and its limitation. When applied to resting-state fMRI data, we find robust evidence of higher-order synergies that are largely invisible to standard functional connectivity analyses. Our approach can also be localized in time, allowing a frame-by-frame analysis of how the distributions of redundancies and synergies change over the course of a recording. We find that different ensembles of regions can transiently change from being redundancy-dominated to synergy-dominated and that the temporal pattern is structured in time. These results provide strong evidence that there exists a large space of unexplored structures in human brain data that have been largely missed by a focus on bivariate network connectivity models. This synergistic structure is dynamic in time and likely will illuminate interesting links between brain and behavior. Beyond brain-specific application, the PED provides a very general approach for understanding higher-order structures in a variety of complex systems.

Cholinergic modulation supports dynamic switching of resting state networks through selective DMN suppression.

Brain activity during the resting state is widely used to examine brain organization, cognition and alterations in disease states. While it is known that neuromodulation and the state of alertness impact resting-state activity, neural mechanisms behind such modulation of resting-state activity are unknown. In this work, we used a computational model to demonstrate that change in excitability and recurrent connections, due to cholinergic modulation, impacts resting-state activity. The results of such modulation in the model match closely with experimental work on direct cholinergic modulation of Default Mode Network (DMN) in rodents. We further extended our study to the human connectome derived from diffusion-weighted MRI. In human resting-state simulations, an increase in cholinergic input resulted in a brain-wide reduction of functional connectivity. Furthermore, selective cholinergic modulation of DMN closely captured experimentally observed transitions between the baseline resting state and states with suppressed DMN fluctuations associated with attention to external tasks. Our study thus provides insight into potential neural mechanisms for the effects of cholinergic neuromodulation on resting-state activity and its dynamics.

Test-retest reliability and time-of-day variations of perfusion imaging at rest and during a vigilance task.

Arterial spin-labeled perfusion and blood oxygenation level-dependent functional MRI are indispensable tools for noninvasive human brain imaging in clinical and cognitive neuroscience, yet concerns persist regarding the reliability and reproducibility of functional MRI findings. The circadian rhythm is known to play a significant role in physiological and psychological responses, leading to variability in brain function at different times of the day. Despite this, test-retest reliability of brain function across different times of the day remains poorly understood. This study examined the test-retest reliability of six repeated cerebral blood flow measurements using arterial spin-labeled perfusion imaging both at resting-state and during the psychomotor vigilance test, as well as task-induced cerebral blood flow changes in a cohort of 38 healthy participants over a full day. The results demonstrated excellent test-retest reliability for absolute cerebral blood flow measurements at rest and during the psychomotor vigilance test throughout the day. However, task-induced cerebral blood flow changes exhibited poor reliability across various brain regions and networks. Furthermore, reliability declined over longer time intervals within the day, particularly during nighttime scans compared to daytime scans. These findings highlight the superior reliability of absolute cerebral blood flow compared to task-induced cerebral blood flow changes and emphasize the importance of controlling time-of-day effects to enhance the reliability and reproducibility of future brain imaging studies.

Functionality of arousal-regulating brain circuitry at rest predicts human cognitive abilities.

Arousal state is regulated by subcortical neuromodulatory nuclei, such as locus coeruleus, which send wide-reaching projections to cortex. Whether higher-order cortical regions have the capacity to recruit neuromodulatory systems to aid cognition is unclear. Here, we hypothesized that select cortical regions activate the arousal system, which, in turn, modulates large-scale brain activity, creating a functional circuit predicting cognitive ability. We utilized the Human Connectome Project 7T functional magnetic resonance imaging dataset (n = 149), acquired at rest with simultaneous eye tracking, along with extensive cognitive assessment for each subject. First, we discovered select frontoparietal cortical regions that drive large-scale spontaneous brain activity specifically via engaging the arousal system. Second, we show that the functionality of the arousal circuit driven by bilateral posterior cingulate cortex (associated with the default mode network) predicts subjects' cognitive abilities. This suggests that a cortical region that is typically associated with self-referential processing supports cognition by regulating the arousal system.

Prediction of individual brain age using movie and resting-state fMRI.

Brain age is a promising biomarker for predicting chronological age based on brain imaging data. Although movie and resting-state functional MRI techniques have attracted much research interest for the investigation of brain function, whether the 2 different imaging paradigms show similarities and differences in terms of their capabilities and properties for predicting brain age remains largely unexplored. Here, we used movie and resting-state functional MRI data from 528 participants aged from 18 to 87 years old in the Cambridge Centre for Ageing and Neuroscience data set for functional network construction and further used elastic net for age prediction model building. The connectivity properties of movie and resting-state functional MRI were evaluated based on the connections supporting predictive model building. We found comparable predictive abilities of movie and resting-state connectivity in estimating brain age of individuals, as evidenced by correlation coefficients of 0.868 and 0.862 between actual and predicted age, respectively. Despite some similarities, notable differences in connectivity properties were observed between the predictive models using movie and resting-state functional MRI data, primarily involving components of the default mode network. Our results highlight that both movie and resting-state functional MRI are effective and promising techniques for predicting brain age. Leveraging its data acquisition advantages, such as improved child and patient compliance resulting in reduced motion artifacts, movie functional MRI is emerging as an important paradigm for studying brain function in pediatric and clinical populations.

Gender differences in the functional language networks at birth: a resting-state fNIRS study.

Numerous studies reported inconsistent results concerning gender influences on the functional organization of the brain for language in children and adults. However, data for the gender differences in the functional language networks at birth are sparse. Therefore, we investigated gender differences in resting-state functional connectivity in the language-related brain regions in newborns using functional near-infrared spectroscopy. The results revealed that female newborns demonstrated significantly stronger functional connectivities between the superior temporal gyri and middle temporal gyri, the superior temporal gyri and the Broca's area in the right hemisphere, as well as between the right superior temporal gyri and left Broca's area. Nevertheless, statistical analysis failed to reveal functional lateralization of the language-related brain areas in resting state in both groups. Together, these results suggest that the onset of language system might start earlier in females, because stronger functional connectivities in the right brain in female neonates were probably shaped by the processing of prosodic information, which mainly constitutes newborns' first experiences of speech in the womb. More exposure to segmental information after birth may lead to strengthened functional connectivities in the language system in both groups, resulting in a stronger leftward lateralization in males and a more balanced or leftward dominance in females.

MRI resting-state signature of the propensity to experience meaningful coincidences: a functional coupling analysis.

The propensity to experience meaningful patterns in random arrangements and unrelated events shows considerable interindividual differences. Reduced inhibitory control (over sensory processes) and decreased working memory capacities are associated with this trait, which implies that the activation of frontal as well as posterior brain regions may be altered during rest and working memory tasks. In addition, people experiencing more meaningful coincidences showed reduced gray matter of the left inferior frontal gyrus (IFG), which is linked to the inhibition of irrelevant information in working memory and the control and integration of multisensory information. To study deviations in the functional connectivity of the IFG with posterior associative areas, the present study investigated the fMRI resting state in a large sample of n = 101 participants. We applied seed-to-voxel analysis and found that people who perceive more meaningful coincidences showed negative functional connectivity of the left IFG (i.e. pars triangularis) with areas of the left posterior associative cortex (e.g. superior parietal cortex). A data-driven multivoxel pattern analysis further indicated that functional connectivity of a cluster located in the right cerebellum with a cluster including parts of the left middle frontal gyrus, left precentral gyrus, and the left IFG (pars opercularis) was associated with meaningful coincidences. These findings add evidence to the neurocognitive foundations of the propensity to experience meaningful coincidences, which strengthens the idea that deviations of working memory functions and inhibition of sensory and motor information explain why people experience more meaning in meaningless noise.

Get out of my head: social evaluative brain states carry over into post-feedback rest and influence remembering how others view us.

Learning how others perceive us helps us tune our behavior to form adaptive relationships. But which perceptions stick with us? And when in the learning process are they codified in memory? We leveraged a popular television series-The Office-to answer these questions. Prior to their functional magnetic resonance imaging (fMRI) session, viewers of The Office reported which characters they identified with, as well as which characters they perceived another person (i.e. counterpart) was similar to. During their fMRI scan, participants found out which characters other people thought they and the counterpart were like, and also completed rest scans. Participants remembered more feedback inconsistent with their self-views (vs. views of the counterpart). Although neural activity while encoding self-inconsistent feedback did not meaningfully predict memory, returning to the inconsistent self feedback during subsequent rest did. During rest, participants reinstated neural patterns engaged while receiving self-inconsistent feedback in the dorsomedial prefrontal cortex (DMPFC). DMPFC reinstatement also quadratically predicted self-inconsistent memory, with too few or too many reinstatements compromising memory performance. Processing social feedback during rest may impact how we remember and integrate the feedback, especially when it contradicts our self-views.

Frontal pole-precuneus connectivity is associated with a discrepancy between self-rated and observer-rated depression severity in mood disorders: a resting-state functional magnetic resonance imaging study.

Discrepancies in self-rated and observer-rated depression severity may underlie the basis for biological heterogeneity in depressive disorders and be an important predictor of outcomes and indicators to optimize intervention strategies. However, the neural mechanisms underlying this discrepancy have been understudied. This study aimed to examine the brain networks that represent the neural basis of the discrepancy between self-rated and observer-rated depression severity using resting-state functional MRI. To examine the discrepancy between self-rated and observer-rated depression severity, self- and observer-ratings discrepancy (SOD) was defined, and the higher and lower SOD groups were selected from depressed patients as participants showing extreme deviation. Resting-state functional MRI analysis was performed to examine regions with significant differences in functional connectivity in the two groups. The results showed that, in the higher SOD group compared to the lower SOD group, there was increased functional connectivity between the frontal pole and precuneus, both of which are subregions of the default mode network that have been reported to be associated with ruminative and self-referential thinking. These results provide insight into the association of brain circuitry with discrepancies between self- and observer-rated depression severity and may lead to more treatment-oriented diagnostic reclassification in the future.

Brain state limits propagation of neural signals in laminar cortical circuits.

Our perception of the environment relies on the efficient propagation of neural signals across cortical networks. During the time course of a day, neural responses fluctuate dramatically as the state of the brain changes to possibly influence how electrical signals propagate across neural circuits. Despite the importance of this issue, how patterns of spiking activity propagate within neuronal circuits in different brain states remains unknown. Here, we used multielectrode laminar arrays to reveal that brain state strongly modulates the propagation of neural activity across the layers of early visual cortex (V1). We optogenetically induced synchronized state transitions within a group of neurons and examined how far electrical signals travel during wakefulness and rest. Although optogenetic stimulation elicits stronger neural responses during wakefulness relative to rest, signals propagate only weakly across the cortical column during wakefulness, and the extent of spread is inversely related to arousal level. In contrast, the light-induced population activity vigorously propagates throughout the entire cortical column during rest, even when neurons are in a desynchronized wake-like state prior to light stimulation. Mechanistically, the influence of global brain state on the propagation of spiking activity across laminar circuits can be explained by state-dependent changes in the coupling between neurons. Our results impose constraints on the conclusions of causal manipulation studies attempting to influence neural function and behavior, as well as on previous computational models of perception assuming robust signal propagation across cortical layers and areas.

Brain-wide functional connectivity of face patch neurons during rest.

The brain is a highly organized, dynamic system whose network architecture is often assessed through resting functional magnetic resonance imaging (fMRI) functional connectivity. The functional interactions between brain areas, including those observed during rest, are assumed to stem from the collective influence of action potentials carried by long-range neural projections. However, the contribution of individual neurons to brain-wide functional connectivity has not been systematically assessed. Here we developed a method to concurrently measure and compare the spiking activity of local neurons with fMRI signals measured across the brain during rest. We recorded spontaneous activity from neural populations in cortical face patches in the macaque during fMRI scanning sessions. Individual cells exhibited prominent, bilateral coupling with fMRI fluctuations in a restricted set of cortical areas inside and outside the face patch network, partially matching the pattern of known anatomical projections. Within each face patch population, a subset of neurons was positively coupled with the face patch network and another was negatively coupled. The same cells showed inverse correlations with distinct subcortical structures, most notably the lateral geniculate nucleus and brainstem neuromodulatory centers. Corresponding connectivity maps derived from fMRI seeds and local field potentials differed from the single unit maps, particularly in subcortical areas. Together, the results demonstrate that the spiking fluctuations of neurons are selectively coupled with discrete brain regions, with the coupling governed in part by anatomical network connections and in part by indirect neuromodulatory pathways.

Ca2+ leak through ryanodine receptor 1 regulates thermogenesis in resting skeletal muscle.

Mammals rely on nonshivering thermogenesis (NST) from skeletal muscle so that cold temperatures can be tolerated. NST results from activity of the sarcoplasmic reticulum (SR) Ca2+ pump in skeletal muscle, but the mechanisms that regulate this activity are unknown. Here, we develop a single-fiber assay to investigate the role of Ca2+ leak through ryanodine receptor 1 (RyR1) to generate heat at the SR Ca2+ pump in resting muscle. By inhibiting a subpopulation of RyR1s in a single-fiber preparation via targeted delivery of ryanodine through transverse tubules, we achieve in-preparation isolation of RyR1 Ca2+ leak. This maneuver provided a critical increase in signal-to-noise of the SR-temperature-sensitive dye ER thermoyellow fluorescence signal from the fiber to allow detection of SR temperature changes as either RyR1 or SR Ca2+ pump activity was altered. We found that RyR1 Ca2+ leak raises cytosolic [Ca2+] in the local vicinity of the SR Ca2+ pump to amplify thermogenesis. Furthermore, gene-dose-dependent increases in RyR1 leak in RYR1 mutant mice result in progressive rises in leak-dependent heat, consistent with raised local [Ca2+] at the SR Ca2+ pump via RyR1 Ca2+ leak. We also show that basal RyR Ca2+ leak and the heat generated by the SR Ca2+ pump in the absence of RyR Ca2+ leak is greater in fibers from mice than from toads. The distinct function of RyRs and SR Ca2+ pump in endothermic mammals compared to ectothermic amphibians provides insights into the mechanisms by which mammalian skeletal muscle achieves thermogenesis at rest.

Cardiopulmonary haemodynamics in Tibetans and Han Chinese during rest and exercise.

During sea-level exercise, blood flow through intrapulmonary arteriovenous anastomoses (IPAVA) in humans without a patent foramen ovale (PFO) is negatively correlated with pulmonary pressure. Yet, it is unknown whether the superior exercise capacity of Tibetans well adapted to living at high altitude is the result of lower pulmonary pressure during exercise in hypoxia, and whether their cardiopulmonary characteristics are significantly different from lowland natives of comparable ancestry (e.g. Han Chinese). We found a 47% PFO prevalence in male Tibetans (n = 19) and Han Chinese (n = 19) participants. In participants without a PFO (n = 10 each group), we measured heart structure and function at rest and peak oxygen uptake ( V ̇ O 2 peak ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{peak}}}}$ ), peak power output ( W ̇ p e a k ${{\dot{W}}_{peak}}$ ), pulmonary artery systolic pressure (PASP), blood flow through IPAVA and cardiac output ( Q ̇ T ${{\dot{Q}}_{\mathrm{T}}} $ ) at rest and during recumbent cycle ergometer exercise at 760 Torr (SL) and at 410 Torr (ALT) barometric pressure in a pressure chamber. Tibetans achieved a higher W peak ${W}_{\textit{peak}}$ than Han, and a higher V ̇ O 2 peak ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{peak}}}}$ at ALT without differences in heart rate, stroke volume or Q ̇ T ${{\dot{Q}}_{\mathrm{T}}} $ . Blood flow through IPAVA was generally similar between groups. Increases in PASP and total pulmonary resistance at ALT were comparable between the groups. There were no differences in the slopes of PASP plotted as a function of Q ̇ T ${{\dot{Q}}_{\mathrm{T}}} $ during exercise. In those without PFO, our data indicate that the superior aerobic exercise capacity of Tibetans over Han Chinese is independent of cardiopulmonary features and more probably linked to differences in local muscular oxygen extraction. KEY POINTS: Patent foramen ovale (PFO) prevalence was 47% in Tibetans and Han Chinese living at 2 275 m. Subjects with PFO were excluded from exercise studies. Compared to Han Chinese, Tibetans had a higher peak workload with acute compression to sea level barometric pressure (SL) and acute decompression to 5000 m altitude (ALT). Comprehensive cardiac structure and function at rest were not significantly different between Han Chinese and Tibetans. Tibetans and Han had similar blood flow through intrapulmonary arteriovenous anastomoses (IPAVA) during exercise at SL. Peak pulmonary artery systolic pressure (PASP) and total pulmonary resistance were different between SL and ALT, with significantly increased PASP for Han compared to Tibetans at ALT. No differences were observed between groups at acute SL and ALT.

Resting membrane potential and intracellular [Na+] at rest, during fatigue and during recovery in rat soleus muscle fibres in situ.

Large trans-sarcolemmal ionic shifts occur with fatiguing exercise or stimulation of isolated muscles. However, it is unknown how resting membrane potential (EM) and intracellular sodium concentration ([Na+]i) change with repeated contractions in living mammals. We investigated (i) whether [Na+]i (peak, kinetics) can reveal changes of Na+-K+ pump activity during brief or fatiguing stimulation and (ii) how resting EM and [Na+]i change during fatigue and recovery of rat soleus muscle in situ. Muscles of anaesthetised rats were stimulated with brief (10 s) or repeated tetani (60 Hz for 200 ms, every 2 s, for 30 s or 300 s) with isometric force measured. Double-barrelled ion-sensitive microelectrodes were used to quantify resting EM and [Na+]i. Post-stimulation data were fitted using polynomials and back-extrapolated to time zero recovery. Mean pre-stimulation resting EM (layer 2-7 fibres) was -71 mV (surface fibres were more depolarised), and [Na+]i was 14 mM. With deeper fibres, 10 s stimulation (2-150 Hz) increased [Na+]i to 38-46 mM whilst simultaneously causing hyperpolarisations (7.3 mV for 2-90 Hz). Fatiguing stimulation for 30 s or 300 s led to end-stimulation resting EM of -61 to -53 mV, which recovered rapidly (T1/2, 8-22 s). Mean end-stimulation [Na+]i increased to 86-101 mM with both fatigue protocols and the [Na+]i recovery time-course (T1/2, 21-35 s) showed no difference between protocols. These combined findings suggest that brief stimulation hyperpolarises the resting EM, likely via maximum Na+-induced stimulation of the Na+-K+ pump. Repeated tetani caused massive depolarisation and elevations of [Na+]i that together lower force, although they likely interact with other factors to cause fatigue. [Na+]i recovery kinetics provided no evidence of impaired Na+-K+ pump activity with fatigue. KEY POINTS: It is uncertain how resting membrane potential, intracellular sodium concentration ([Na+]i), and sodium-potassium (Na+-K+) pump activity change during repeated muscle contractions in living mammals. For rat soleus muscle fibres in situ, brief tetanic stimulation for 10 s led to raised [Na+]i, anticipated to evoke maximal Na+-induced stimulation of the Na+-K+ pump causing an immediate hyperpolarisation of the sarcolemma. More prolonged stimulation with repeated tetanic contractions causes massive elevations of [Na+]i, which together with large depolarisations (via K+ disturbances) likely reduce force production. These effects occurred without impairment of Na+-K+ pump function. Together these findings suggest that rapid activation of the Na+-K+ pump occurs with brief stimulation to maintain excitability, whereas more prolonged stimulation causes rundown of the trans-sarcolemmal K+ gradient (hence depolarisation) and Na+ gradient, which in combination can impair contraction to contribute to fatigue in living mammals.