Light activates visual pigments and downstream G protein signalling cascades in rod and cone photoreceptor cells. Timely response inactivation requires visual pigment inactivation by arrestin and restoration of cellular cGMP by guanylate cyclase activating proteins (GCAPs). Using GCAP-deficient mice, we previously showed that apo-opsin produces photoresponse-like signals. To investigate the inactivation of apo-opsin by arrestin, we generated GCAP-deficient mice lacking arrestin (Gcaps[-/-]Arr1[-/-]). Using ex vivo electroretinography, we found that the dim flash responses of Gcaps[-/-]Arr1[-/-] rods were ∼100-fold slower than those of control Gcaps[-/-] rods. As a result, even brief dim light exposure during sample preparation resulted in prolonged rod saturation, so that only cone responses were initially observed in Gcaps[-/-]Arr1[-/-] retinas. In contrast, rod responses were observed immediately when Gcaps[-/-]Arr1[-/-] retinas were dissected under infrared light to avoid rod photoactivation. Microspectrophotometry showed that the rate of production and decay of the active rhodopsin photoproduct, Meta II, was comparable between Gcaps[-/-] and Gcaps[-/-]Arr1[-/-] rods. However, the rate of photoproduct Meta III decay was 3-fold slower in Gcaps[-/-]Arr1[-/-] rods, supporting a role for arrestin in accelerating the decay of photoactivated rhodopsin. To evaluate the effect of rod saturation on cone responses, we compared photopic cone responses from wild-type and Gnat1[-/-] retinas lacking rod signalling. Cone photoresponses were larger and more sensitive in wild-type retinas, suggesting the potentiation of cone function by the saturation of rod phototransduction. Together, our results demonstrate that the very slow deactivation of rod responses in Gcaps[-/-]Arr1[-/-] retinas might result in selective and potentiated cone signalling in darkness. KEY POINTS: Arrestin and guanylate cyclase activating proteins (GCAPs) play an important role in inactivation of the phototransduction cascade and termination of the light response in rod photoreceptors. The deletion of arrestin and GCAPs results in ∼100-fold slower dim flash responses in rods so that even brief dim light exposure produces saturation of the rods for tens of minutes. The decay of the rhodopsin photoactivated intermediate Meta III was slowed down 3-fold by the deletion of arrestin in Gcaps[-/-] rods, supporting a role for arrestin in accelerating the decay of photoactivated rhodopsin. Cone photoresponses were smaller and less sensitive in retinas lacking rod signalling, suggesting potentiation of cone function by the saturation of rod phototransduction. Together, these results demonstrate that the very slow deactivation of rod responses in arrestin- and GCAP-deficient retinas can result in selective and potentiated cone signalling in darkness.

Effective interaction with complex environments relies on the integration of somatosensory and visual signals into coherent spatial representations of the body in external space. When these signals conflict, as during goal-directed hand movements under mirror-reversed visual feedback, motor performance deteriorates. Despite this conflict, the visual mapping between the hand and the environment remains preserved, and visual information typically facilitates motor performance, whereas tactile feedback impairs it. Here, we tested whether providing tactile input with explicit spatial structure renders visuo-somatosensory integration functionally relevant and stabilizes motor behaviour under sensory conflict. Healthy adults performed a mirror-tracing task, tracing a shape's contour with the index finger while tactile feedback conveyed either motion-related cues alone or additional discrete spatial landmarks aligned with the outline. Spatially structured tactile cues preserved movement accuracy and smoothness relative to motion-related tactile input, indicating more effective online motor control. EEG source analyses further revealed that, when spatial tactile cues were present, somatosensory activity increased and functional connectivity was largely restricted to sensorimotor areas. In contrast, when tactile cues did not convey spatial information, sensory conflict was associated with widespread reductions in cortical activity, including in somatosensory and parietal areas, and with a diffuse functional connectivity pattern consistent with elevated sensorimotor uncertainty. Together, these findings indicate that providing spatial structure to tactile input can mitigate the detrimental effects of visuo-somatosensory conflict on motor performance and support more efficient integration of visual and somatosensory information during action. KEY POINTS: Accurate movements depend on combining visual and somatosensory information to estimate the position of the body in external space. When these signals conflict, motor performance usually deteriorates and tactile feedback can become disruptive. We tested whether tactile feedback becomes useful for movement control when it provides explicit spatial landmarks, rather than only motion-related information from finger-surface contact. During a tracing task under visuo-somatosensory conflict, spatially structured tactile cues preserved movement accuracy and smoothness compared with when touch conveyed motion cues alone. Spatial tactile cues were associated with increased somatosensory cortical activity and a more spatially restricted pattern of functional coupling within sensorimotor networks, whereas their absence was associated with reduced somatosensory and parietal activity and a more distributed connectivity profile. Spatial structure in touch promotes multisensory integration, helping stabilize motor control under sensory conflict and highlighting how tactile cue design may improve sensorimotor function.

Breathing modulates cortical activities and cognitive functions, but the neurophysiological basis of the brain-breathing relationship is incompletely understood. We aimed to determine whether breathing at rest evokes scalp EEG-detectable potentials (as inspiratory occlusions do in the respiratory-related evoked potential model), whether these signatures persist during sleep and whether they are linked to sleep-organizing processes. We examined recordings from 729 participants in the Cleveland Family Study to identify cortical activity time-locked to inspiration during wakefulness and sleep, with a median of 7871 respiratory cycles analysed per participant. Breathing-related cortical responses were assessed in the time and frequency domains. Quiet breathing evoked a reproducible sequence of cortical potentials during wakefulness, including a pre-inspiratory component ∼300 ms before inspiration and several post-inspiratory peaks ≤3 s after. These responses persisted during non-rapid eye movement stages 2 and 3 (N2 and N3) and rapid eye movement (REM) sleep, in which they showed simplified morphology resembling K-complexes and co-occurred with increased spindle-range activity. The pattern was present irrespective of body mass index and was qualitatively unchanged in the 203 participants free of sleep-disordered breathing, arguing against obesity or sleep apnoea as its origin. Scalp EEG can detect breathing-evoked potentials during quiet breathing in awake humans. These potentials persist during sleep with a morphology that resembles cortical activities previously associated with sleep stabilization. Further studies are needed to determine the physiological and clinical significance of these observations in health and disease. KEY POINTS: Spontaneous breathing at rest evokes reproducible breathing-related cortical potentials detectable with scalp EEG. These potentials persist during sleep. They are associated with time-locked modulation of cortical oscillatory activity.

The Western diet (WD) is associated with skeletal muscle mitochondrial dysfunction, impaired oxidative metabolism, redox imbalance and inflammation. Emerging evidence suggests that gut microbiome-derived metabolites contribute to host metabolic regulation, yet the role of Bacteroidota-derived glycine lipids in skeletal muscle remains unknown. This study tested whether two structurally related bacterial glycine lipids, lipid 654 (L654) and lipid 1256 (L1256), regulate skeletal muscle mitochondrial remodelling in WD-fed Ldlr[-/-] mice. Male Ldlr[-/-] mice were fed a normal chow or WD and administered L654, L1256 or vehicle control for 8 weeks. Skeletal muscle glycine lipid accumulation, mitochondrial respiration, mitochondrial biogenesis markers, mitophagy-associated signalling, inflammatory and antioxidant proteins and muscle fibre cross-sectional area were assessed. L1256, but not L654, accumulated in skeletal muscle. Functionally, L1256 selectively enhanced fatty acid-supported mitochondrial respiration and increased respiratory control ratio. L1256 also increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), mitochondrial transcription factor A (TFAM) and electron transport chain complexes IV and V, suggesting enhanced oxidative mitochondrial remodelling. In parallel, L1256 altered markers associated with mitochondrial quality control signalling, increased nuclear factor erythroid 2-related factor 2 (Nrf2), manganese superoxide dismutase (MnSOD) and glutathione peroxidase 4 (GPX4), suppressed nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling and preserved muscle fibre cross-sectional area. L654 upregulated mitochondrial biogenesis transcripts without altering protein abundance. These findings identify L1256 as a microbiome-derived lipid retained within skeletal muscle that enhanced oxidative metabolism and preserved mitochondrial quality signalling under metabolic stress. The data suggest that tissue retention of bacterial lipids may be an important determinant of skeletal muscle mitochondrial adaptation. KEY POINTS: Western diet contributes to skeletal muscle mitochondrial dysfunction, impaired oxidative metabolism and chronic inflammation. Microbiome-derived glycine lipids are bioactive molecules from Bacteroidota species, but their effects on skeletal muscle mitochondrial metabolism remain poorly understood. Bacteroidota-derived glycine lipids produce distinct skeletal muscle responses in Western diet-fed Ldlr[-] [/] [-] mice, where lipid 1256 accumulated in skeletal muscle and enhanced fatty acid-supported respiration, whereas lipid 654 primarily induced transcriptional responses without detectable tissue accumulation. Lipid 1256 increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), mitochondrial transcription factor A (TFAM) and complexes IV and V of the electron transport chain together with restoration of Parkin, nuclear factor erythroid 2-related factor 2 (Nrf2), manganese superoxide dismutase (MnSOD) and glutathione peroxidase 4 (GPX4) while reducing nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interleukin 6 (IL-6) expression and preserving skeletal muscle cross-sectional area. These findings suggest that gut microbiome-derived glycine lipids are regulators of skeletal muscle mitochondrial metabolism, and that tissue retention of bacterial lipids influences mitochondrial oxidative remodelling during metabolic stress.

Several ion channels belonging to the large TRP family are activated either by increases or decreases in temperature, but it is unclear which of these potential thermosensors actually determine the sensation of temperature in the non-noxious thermal range, from approximately 20°C to approximately 40°C. Here we show that below 33°C, male cool preference is determined by TRPM8, whereas warm preference is mainly influenced by TRPM2. Female thermal preference is shifted to warmer temperatures by around 4°C when compared to males, consistent with warm shifts in the female activation ranges of both TRPM8 and TRPM2. Removal of testosterone causes a shift in male thermal preference towards female preference; conversely, female thermal preference is shifted towards male by injection of testosterone. Removing or injecting female hormones had no effect on thermal preference. We conclude that TRPM8 and TRPM2 are the main thermal detectors in the non-noxious range, and that testosterone is an important determinant of the difference in preference between males and females. KEY POINTS: In many mammals, including humans, females show a preference for warmer temperatures than males. Deletion of the trpm2 gene, coding for the warmth sensor TRPM2 in male mice shifted preference to warmer temperatures, showing that TRPM2 repels males from warm temperatures. Deletion or block of the cool sensor trpm8 in both males and females shifted preference to cooler temperatures, showing that TRPM8 repels mice from cooler temperatures. Deletion or block of trpv1 had no effect on non-noxious thermal preference. The results can be explained by a warm shift in females of activation ranges of both TRPM8 and TRPM2 when compared to males. Reducing testosterone in males caused a shift towards the female warm preference. Administering testosterone to females caused a shift towards the male cool preference. Female sex hormones had no effect. We conclude that TRPM8 and TRPM2 determine non-noxious thermal preference. Testosterone is an important determinant of the differences in thermal preference between males and females.

During early development, temporoammonic (TA) input from the entorhinal cortex provides major excitatory drive to the CA1 hippocampus, yet whether this excitation is balanced by feedforward inhibition remains unclear. Using whole-cell recordings from CA1 pyramidal cells (PCs) in intact hippocampi, we found that TA stimulation reliably evoked monosynaptic excitatory postsynaptic currents (EPSCs) from birth, whereas inhibitory postsynaptic currents (IPSCs) were frequently absent or severely delayed during the first postnatal week. The delay between evoked EPSCs and IPSCs shortened with age, narrowing the temporal integration window. Similar dynamics occurred during early sharp waves in vivo, where IPSC bursts followed EPSC bursts by tens of milliseconds, with this delay decreasing over development. Using paired recordings from fast-spiking interneurons (FS-INs) and PCs, we identified two mechanisms underlying delayed feedforward inhibition during the first postnatal week: (i) low FS-IN→PC connectivity, and (ii) a weak monosynaptic entorhinal cortex (EC) drive to FS-INs. By the end of the second week, EC inputs to FS-INs surpassed those to PCs. Together, these findings reveal that feedforward inhibition in the EC-CA1 circuit is delayed, creating a broad temporal window for synaptic integration during the neonatal period. We propose that this extended window supports global bursting, with progressive inhibition maturation contributing to the switch toward sparse coding. KEY POINTS: Feedforward inhibition upon entorhinal input is developmentally delayed, creating a broad temporal integration window in the neonatal CA1 hippocampus. Weak entorhinal drive to CA1 fast-spiking interneurons is the primary cause of delayed feedforward inhibition in neonates. Delayed inhibition is a consistent feature of CA1 early sharp waves in vivo. Maturation of feedforward inhibition may drive the switch from global 'bursting' to sparse coding.

Skeletal muscle mass reflects the balance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB). In healthy humans, a decline in MPS is recognized as the primary driver of muscle disuse-atrophy, with limited contribution from increased MPB. However, disuse-induced declines in protein pool size directly influence the calculation of muscle protein turnover. We examined how accounting for changes in protein pool size impacted MPS and MPB rates assessed using deuterium oxide in healthy humans. Fourteen females (21 ± 2 years of age; 24.5 ± 3.0 kg/m[2]) had one leg subjected to 10 days of unloading, whilst the contralateral leg underwent loading in the form of resistance exercise. Steady-state and non-steady-state modelling approaches were used to calculate MPS and MPB rates. In response to unloading, there was a significant decline in MRI-derived vastus lateralis volume, and thus non-steady-state equations were applied exclusively to this limb. Regardless of whether steady-state or non-steady-state approaches were applied, mixed MPS rates were significantly reduced in response to unloading, whilst mixed MPB rates remained unchanged. Notably, unloading elicited protein fraction-specific responses, whereby myofibrillar MPS rates declined significantly whilst sarcoplasmic MPS rates were unaltered. We also found that in response to loading, mixed, myofibrillar and sarcoplasmic MPS rates remained unchanged compared with basal rates. Our findings show that unilateral unloading in females induces muscle disuse-atrophy driven by reductions in MPS rates, rather than by elevations in MPB rates, a response that was protein fraction specific and preserved irrespective of whether steady-state or non-steady-state equations were used. KEY POINTS: In humans, short-term muscle disuse-atrophy is generally attributed to reduced muscle protein synthesis (MPS) rates, but integrated tracer approaches to measure protein turnover can be influenced by changes in protein pool size. We used deuterated water ([2]H2O) to measure rates of MPS and muscle protein breakdown (MPB) during 10 days of unilateral unloading in young, healthy females. Steady-state and non-steady-state equations were applied to account for changes in the protein pool size that occur with disuse-atrophy. Unloading reduced muscle size and MPS rates, whereas MPB rates were unchanged. Accounting for the change in the protein pool size using non-steady-state equations did not alter this conclusion. These findings refine our interpretation of the mechanisms driving muscle disuse-atrophy in healthy humans that might differ from more severe clinical or preclinical scenarios of muscle wasting.

The endocannabinoid system is important for pain modulation, whereas its utilization for analgesia is restricted due to the limited efficacy of cannabinoids and side effects. Cannabinoid receptor 1 (CB1) in the spinal dorsal horn provides presynaptic inhibition of nociceptive neurotransmission. CB1 regulatory protein, SH3-containing GRB2-like protein 3-interacting protein 1 (SGIP1), controls its functional availability, while its role in pain modulation remains poorly understood. We examined the role of SGIP1 in nociceptive signalling under control and inflammatory conditions, using behavioural tests, patch-clamp recordings in spinal cord slices and calcium imaging of dorsal root ganglion (DRG) neurons. SGIP1 knockout (KO) mice of both sexes showed a selective reduction in heat sensitivity, with no change in mechanical withdrawal threshold. Following carrageenan-induced inflammation, both wild-type (WT) and KO mice developed mechanical allodynia, whereas thermal hyperalgesia in KO mice reached only the control values of WT mice. Intrathecal application of cannabinoid agonist WIN55,212-2 produced analgesia in both WT and KO mice, with enhanced effect of the lower dose (5 µg vs. 10 µg) in KO animals. WIN55,212-2 (1 µM) in slices from inflamed SGIP1 KO mice induced significantly greater reduction in miniature excitatory postsynaptic current (mEPSC) frequency, without amplitude change, indicating enhanced presynaptic inhibition in KO mice. These enhanced inhibitory effects of WIN55,212-2 in KO mice after inflammation were further demonstrated in DRG cultured neurons. Our results establish SGIP1 as a state-dependent negative modulator of CB1 function and identify SGIP1-CB1 interaction as a candidate target for selective enhancement of cannabinoid analgesia under inflammatory pain conditions. KEY POINTS: SGIP1 deletion reduced basal heat sensitivity in mice of both sexes without altering mechanical threshold, suggesting a sex-independent role in thermal nociception. Inflammation increased mechanical and thermal sensitivity in both SGIP1 KO and WT mice, whereas the increased thermal sensitivity of KO reached only the baseline levels of WT mice. Intrathecal cannabinoid agonist WIN55,212-2 produced analgesia in both genotypes, with a bigger effect of the lower dose (5 vs. 10 µg) in SGIP1 KO mice. WIN55,212-2 induced strong inhibition of miniature excitatory postsynaptic current (mEPSC) frequency in dorsal horn neurons that was selectively potentiated by SGIP1 deletion under inflammatory, but not control, conditions, consistent with a presynaptic locus of action. These effects were further confirmed by calcium imaging in DRG cultured neurons. SGIP1 acts as an inflammation-dependent presynaptic CB1 modulator of spinal nociceptive transmission, positioning the SGIP1-CB1 interaction as a candidate target for selective enhancement of cannabinoid analgesia.

Localized application of heat is used therapeutically to reduce skeletal muscle pain and improve mobility and recovery in various populations. The efficacy of this thermal therapy is considered to be partly a result of increased blood flow in the skeletal muscle directly exposed to heat (i.e. thermal hyperaemia). However, the mechanisms mediating thermal hyperaemia in skeletal muscle of humans remain poorly understood. Voltage-dependent potassium (KV) channels play a pivotal role linking upstream vasodilatory signals to electro-mechanical mechanisms that mediate vascular smooth muscle relaxation. Therefore, the present study aimed to test the hypothesis that KV channel antagonism would attenuate thermal hyperaemia in skeletal muscle of humans. We used microdialysis to directly measure local blood flow via the ethanol washout technique at two sites in the vastus lateralis muscle of 10 healthy adults (seven women). One probe was perfused with 0.9% saline, whereas the other was perfused with the KV channel antagonist 4-aminopyridine (26.6 mm). Pulsed short-wave diathermy was used to induce deep heating of the vastus lateralis for 90 min. Muscle temperature was measured midway between the microdialysis probes via a thermocouple. Baseline blood flow did not differ between probes (P = 0.684). Pulsed short-wave diathermy increased skeletal muscle temperature through 90 min of heating (P < 0.001). However, the accompanying increase in blood flow at 90 min was attenuated when perfused with 4-aminopyridine (P = 0.0222). These data suggest that KV channels substantially contribute to thermal hyperaemia in skeletal muscle of humans. KEY POINTS: The efficacy of thermal therapy is considered to be partly a result of increased blood flow in the skeletal muscle directly exposed to heat (i.e. thermal hyperaemia). However, the mechanisms mediating thermal hyperaemia in skeletal muscle remain poorly understood. Voltage-dependent potassium (KV) channels play a pivotal role as downstream effectors that mediate vascular smooth muscle relaxation. Therefore, we tested the hypothesis that KV channel antagonism would attenuate thermal hyperaemia in skeletal muscle of humans. Using microdialysis, we found that KV channel antagonism with 4-aminopyridine attenuated thermal hyperaemia at 90 min of localized heating, when this vasodilatory response was the greatest. We identified a critical role for KV channels in human skeletal muscle thermal hyperaemia, establishing these channels as important downstream effectors of this vasodilatory response.

The intrinsic properties of spinal motoneurons support flexible movement, including the maintenance of postural tone. Motoneurons can produce sustained action potential output that outlasts synaptic input, a phenomenon traditionally attributed to persistent inward currents (PICs) mediated by sodium and calcium channels. Using whole-cell patch-clamp electrophysiology, we examined how specific ion channels contribute to PIC maturation and non-linear firing dynamics that allow motoneurons to sustain their output in fast and slow lumbar motoneurons across postnatal development in mice. PIC amplitude and non-linear firing dynamics increased after weight-bearing in fast but not slow motoneurons. Blocking NaV1.6 channels reduced PIC amplitude at both pre- and post-weight-bearing stages, whereas L-type calcium channel blockade only reduced PICs after weight-bearing emerged. However, reducing PIC amplitude by blocking these channels, either individually or in parallel, did not abolish sustained firing hysteresis, which may be because PICs are not yet fully established at this stage of postnatal development. Unexpectedly, activation of muscarinic receptors increased PIC amplitude while promoting adaptive firing dynamics, suggesting that PICs alone do not account for sustained firing hysteresis. We also found that pharmacological manipulation of potassium currents mediated by KCNQ and KV1.2 channels produced substantial changes in firing dynamics. Strikingly, blocking hyperpolarization-activated cyclic nucleotide-gated channels promoted sustained firing dynamics and led to the emergence of self-sustained firing in fast motoneurons. These results indicate that PICs and non-linear firing dynamics mature together and extend the current framework on non-linear firing dynamics of motoneurons to include additional voltage-gated ion channels. KEY POINTS: Persistent inward currents (PICs) and recruitment-derecruitment hysteresis increase in parallel in fast, but not slow, motoneurons following the onset of hindlimb weight-bearing. Increased expression or function of L-type calcium channels may contribute to enhanced PIC amplitude in fast motoneurons after weight-bearing emerges. Blocking NaV1.6 or L-type calcium channels did not reduce sustained firing hysteresis in fast motoneurons. KCNQ channels oppose PICs and, together with KV1.2 channels, shape recruitment-derecruitment asymmetry, thereby modulating firing hysteresis in fast motoneurons. Hyperpolarization-activated cyclic nucleotide-gated channels generate a resting H-current that delays recruitment, modulates firing hysteresis, and prevents the emergence of self-sustained firing in fast motoneurons.