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Aims: This study aimed to characterize the properties of locus coeruleus (LC) noradrenergic neurons in male and female mice. We also sought to investigate sex‐specific differences in membrane properties, action potential generation, and protein expression profiles to understand the mechanisms underlying neuronal excitability variations. Methods: Utilizing a genetic mouse model by crossing Dbhcre knock‐in mice with tdTomato Ai14 transgenic mice, LC neurons were identified using fluorescence microscopy. Neuronal functional properties were assessed using patch‐clamp recordings. Proteomic analyses of individual LC neuron soma was conducted using mass spectrometry to discern protein expression profiles. Data are available via ProteomeXchange with identifier PXD045844. Results: Female LC noradrenergic neurons displayed greater membrane capacitance than those in male mice. Male LC neurons demonstrated greater spontaneous and evoked action potential generation compared to females. Male LC neurons exhibited a lower rheobase and achieved higher peak frequencies with similar current injections. Proteomic analysis revealed differences in protein expression profiles between sexes, with male mice displaying a notably larger unique protein set compared to females. Notably, pathways pertinent to protein synthesis, degradation, and recycling, such as EIF2 and glucocorticoid receptor signaling, showed reduced expression in females. Conclusions: Male LC noradrenergic neurons exhibit higher intrinsic excitability compared to those from females. The discernible sex‐based differences in excitability could be ascribed to varying protein expression profiles, especially within pathways that regulate protein synthesis and degradation. This study lays the groundwork for future studies focusing on the interplay between proteomics and neuronal function examined in individual cells. [ABSTRACT FROM AUTHOR]

Background: Sarcopenia, or age‐dependent decline in muscle force and power, impairs mobility, increasing the risk of falls, institutionalization, co‐morbidity, and premature death. The discovery of adrenoceptors, which mediate the effects of the sympathetic nervous system (SNS) neurotransmitter norepinephrine on specific tissues, sparked the development of sympathomimetics that have profound influence on skeletal muscle mass. However, chronic administration has serious side effects that preclude their use for muscle‐wasting conditions. Interventions that can adjust neurotransmitter release to changing physiological demands depend on understanding how the SNS affects neuromuscular transmission, muscle motor innervation, and muscle mass. Methods: We examined age‐dependent expression of the heart and neural crest derivative 2 (Hand2), a critical transcription factor for SN maintenance, and we tested the possibility that inducing its expression exclusively in sympathetic neurons (SN) will prevent (i) motor denervation, (ii) impaired neuromuscular junction (NMJ) transmission, and (iii) loss of muscle mass and function in old mice. To test this hypothesis, we delivered a viral vector carrying Hand2 expression or an empty vector exclusively in SNs by vein injection in 16‐month‐old C57BL/6 mice that were sacrificed 6 months later. Techniques include RNA‐sequencing, real‐time PCR, genomic DNA methylation, viral vector construct, tissue immunohistochemistry, immunoblot, confocal microscopy, electrophysiology, and in vivo mouse physical performance. Results: Hand2 expression declines throughout life, but inducing its expression increased (i) the number and size of SNs, (ii) muscle sympathetic innervation, (iii) muscle weight and force and whole‐body strength, (iv) myofiber size but not muscle fibre‐type composition, (v) NMJ transmission and nerve‐evoked muscle force, and (vi) motor innervation in old mice. Additionally, the SN controls a set of genes to reduce inflammation and to promote transcription factor activity, cell signalling, and synapse in the skeletal muscle. Hand2 DNA methylation may contribute, at least partially, to gene silencing. Conclusions: Selective expression of Hand2 in the mouse SNs from middle age through old age increases muscle mass and force by (i) regulating skeletal muscle sympathetic and motor innervation; (ii) improving acetylcholine receptor stability and NMJ transmission; (iii) preventing inflammation and myofibrillar protein degradation; (iv) increasing autophagy; and (v) probably enhancing protein synthesis. [ABSTRACT FROM AUTHOR]

Background: Previous studies support beneficial effects of both resistance exercise training (RT) and caloric restriction (CR) on skeletal muscle strength and physical performance. The goal of this study was to determine the effects of adding CR to RT on single-muscle fiber contractility responses to RT in older overweight and obese adults.Methods: We analyzed contractile properties in 1,253 single myofiber from muscle biopsies of the vastus lateralis, as well as physical performance and thigh muscle volume, in 31 older (65-80 years), overweight or obese (body mass index = 27-35 kg/m2) men (n = 19) and women (n = 12) who were randomly assigned to a standardized, progressive RT intervention with CR (RT+CR; n = 15) or without CR (RT; n = 16) for 5 months.Results: Both interventions evoked an increase in force normalized to cross-sectional area (CSA), in type-I and type-II fibers and knee extensor quality. However, these improvements were not different between intervention groups. In the RT group, changes in total thigh fat volume inversely correlated with changes in type-II fiber force (r = -.691; p = .019). Within the RT+CR group, changes in gait speed correlated positively with changes in type-I fiber CSA (r = .561; p = .030). In addition, increases in type-I normalized fiber force were related to decreases in thigh intermuscular fat volume (r = -0.539; p = .038).Conclusion: Single muscle fiber force and knee extensor quality improve with RT and RT+CR; however, CR does not enhance improvements in single muscle fiber contractility or whole muscle in response to RT in older overweight and obese men and women. [ABSTRACT FROM AUTHOR]

Studies in humans and animal models provide compelling evidence for age-related skeletal muscle denervation, which may contribute to muscle fiber atrophy and loss. Skeletal muscle denervation seems relentless; however, long-term, high-intensity physical activity appears to promote muscle reinnervation. Whether 5-month resistance training (RT) enhances skeletal muscle innervation in obese older adults is unknown. This study found that neural cell-adhesion molecule, NCAM+ muscle area decreased with RT and was inversely correlated with muscle strength. NCAM1 and RUNX1 gene transcripts significantly decreased with the intervention. Type I and type II fiber grouping in the vastus lateralis did not change significantly but increases in leg press and knee extensor strength inversely correlated with type I, but not with type II, fiber grouping. RT did not modify the total number of satellite cells, their number per area, or the number associated with specific fiber subtypes or innervated/denervated fibers. Our results suggest that RT has a beneficial impact on skeletal innervation, even when started late in life by sedentary obese older adults. [ABSTRACT FROM AUTHOR]

Abstract: A population of fast muscle fibers from aging mice is dependent on external Ca2+ to maintain tetanic force during repeated contractions. We hypothesized that age-related denervation in muscle fibers plays a role in initiating this contractile deficit, and that prevention of denervation by IGF-1 overexpression would prevent external Ca2+-dependent contraction in aging mice. IGF-1 overexpression in skeletal muscle prevents age-related denervation, and prevented external Ca2+-dependent contraction in this work. To determine if the effects of IGF-1 overexpression are on muscle or nerve, aging mice were injected with a tetanus toxin fragment-C (TTC) fusion protein that targets IGF-1 to spinal cord motor neurons. This treatment prevented external Ca2+-dependent contraction. We also show evidence that injections of the IGF-1-TTC fusion protein prevent age-related alterations to the nerve terminals at the neuromuscular junctions. We conclude that the slow age-related denervation of fast muscle fibers underlies dependence on external Ca2+ to maintain tetanic force in a population of muscle fibers from senescent mice. [Copyright &y& Elsevier]

In this work we hypothesized that denervation in flexor digitorum brevis (FDB) muscle from ageing mice is more extensive than predicted by standard functional and structural assays used in the past. In addition, we asked whether denervation is a fully or partially developed process. Despite the reported alteration in skeletal muscle innervation, the quantification of the extension and magnitude of denervation in ageing rodents has remained elusive. To address these two questions we utilized a combination of electrophysiological and immunohistochemical assays directed to detecting the expression of tetrodotoxin (TTX)-resistant sodium channels (Nav1.5) in FDB muscles from young-adult and senescent mice. Sodium current density measured with the macropatch cell-attached technique did not show significant differences between FDB fibres from young and old mice. The TTX dose–response curve, using the whole cell voltage-clamp technique, showed three populations of fibres in senescent mice, one similar to fibres from young mice (TTX sensitive), another one similar to fibres from experimentally denervated muscle (TTX resistant), and a third group intermediate between these two. Partially and fully denervated fibres added up to approximately 50% of the total number of fibres tested, a number that concurs with the percentage of fibres positive for the Nav1.5 channel by specific immunostaining. [ABSTRACT FROM AUTHOR]

In the present work, we investigate whether changes in excitation-contraction (EC) coupling mode occur in skeletal muscles from ageing mammals by examining the dependence of EC coupling on extracellular Ca2+. Single intact muscle fibres from flexor digitorum brevis muscles from young (2-6 months) and old (23-30 months) mice were subjected to tetanic contractile protocols in the presence and absence of external Ca2+. Contractile experiments in the absence of external Ca2+ show that about half of muscle fibres from old mice are dependent upon external Ca2+ for maintaining maximal tetanic force output, while young fibres are not. Decreased force in the absence of external Ca2+ was not due to changes in charge movement as revealed by whole-cell patch-clamp experiments. Ca2+ transients, measured by fluo-4 fluorescence, declined in voltage-clamped fibres from old mice in the absence of external Ca2+. Similarly, Ca2+ transients declined in parallel with tetanic contractile force in single intact fibres. Examination of inward Ca2+ current and of mRNA and protein assays suggest that these changes in EC coupling mode are not due to shifts in dihydropyridine receptor (DHPR) and/or ryanodine receptor (RyR) isoforms. These results indicate that a change in EC coupling mode occurs in a population of fibres in ageing skeletal muscle, and is responsible for the age-related dependence on extracellular Ca2+. [ABSTRACT FROM AUTHOR]

Despite the multiple effects on mammals during development, the effectiveness of the insulin-like growth factor-1 (IGF-1) to sustain cell function and structure in the brain of senescent mammals is almost completely unknown. To address this issue, we investigated whether the effects of IGF-1 on specific targets are preserved at later stages of life. Voltage-gated Ca&sup2+; channels (VGCC) are well-characterized targets of IGF-1. VGCC regulate membrane excitability and gene transcription along with other functions that have been found to be impaired in the brain of senescent rodents. As the voluntary control of movement has been reported to be altered in the elderly, we investigated the expression, function and responsiveness of high (HVA)- and low-voltage-activated (LVA) Ca&sup2+; channels to IGF-1, using the whole-cell configuration of the patch-clamp and RT-PCR in the specific region of the rat motor cortex that controls hindlimb muscle movement. We detected the expression of α[sub1A], α[sub1B] and α[sub1E] genes encoding the HVA Ca&sup2+; channels P/Q, N and R, respectively, but not α[sub1C], α[sub1D], α[sub1S] encoding the L-type Ca&sup2+; channel in this region of the brain cortex. IGF-1 enhanced Ca&sup2+; channel currents through P/Q- and N-type channels but not significantly through the R-type or LVA channels. IGF-1 enhanced the amplitude but did not modify the voltage dependence of Ca&sup2+; channel currents in young (2- to 4-week-old), young adult (7-month-old) and senescent (28- to 29-month-old) rats. These results support the concept that despite the reported decrease in circulating (liver) and local (central nervous system) production of IGF-1 with ageing, key neuronal targets such as the VGCC remain responsive to the growth factor throughout life. [ABSTRACT FROM AUTHOR]

In the present work we test the hypothesis that sustained transgenic overexpression of insulin-like growth factor-1 (IGF-1) in skeletal muscle prevents age-related decreases in myoplasmic Ca&sup2+; concentration and consequently in specific force in single intact fibres from the flexor digitorum brevis (FDB) muscle from the mouse. Measurements of IGF-1 concentration in FDB muscle showed higher levels in transgenic than in wild-type mice at all ages. The specific tetanic force decreased significantly in single muscle fibres from old (286 ± 22 kPa) compared to young wild-type (455 ± 28 kPa), young transgenic (423 ± 43 kPa), and old transgenic mice (386 ± 15 kPa) (P < 0.05). These results are consistent with measurements in whole FDB muscles. The peak Ca&sup2+; concentration values in response to prolonged stimulation were: 1.47 ± 0.15, 1.70 ± 0.29, 0.97 ± 0.13 and 1.7 ± 0.2 µM, in fibres from young wild-type, young transgenic, old wild-type and old transgenic mice, respectively. The effects of caffeine on FDB fibres support the conclusion that the age-related decline in peak myoplasmic Ca&sup2+; and specific force is not explained by sarcoplasmic reticulum Ca&sup2+; depletion. Immunohistochemistry in muscle cross-sections was performed to determine whether age and/or IGF-1 overexpression induce changes in fibre type composition. The relative percentages of type IIa, IIx and I myosin heavy chain (MHC) isoforms did not change significantly with age or genotype. Therefore, IGF-1 prevents age-related decline in peak intracellular Ca&sup2+; and specific force in a muscle that does not exhibit changes in fibre type composition with senescence. [ABSTRACT FROM AUTHOR]

The locus coeruleus (LC)‐prefrontal cortex (PFC) circuitry is crucial for cognition, planning, posture and mobility. This study examines the role of norepinephrine (NE) in elucidating the neurobiological basis of age‐related cognitive and motor declines. Aged mice exhibited reduced spatial learning, impaired memory, decreased physical endurance, and notable changes in locomotor behavior. The neurochemical foundations of these deficits were investigated through fast‐scan cyclic voltammetry to measure NE release in the PFC and LC, both in vivo and in brain slices. Additionally, oxygen levels were monitored as a proxy for PFC neuronal function, and NE levels were analyzed in the extracellular space via microdialysis and total content in the PFC. Aged mice exhibited a frequency‐dependent increase in NE release in the PFC upon LC stimulation, suggesting alterations in neural responsiveness due to aging. We also recorded slower NE reuptake rates and increased NE content and neuronal activity, indicated by higher oxygen levels and facilitated neuron activation due to membrane depolarization recorded via whole‐cell patch‐clamp. To understand the basis for LC‐driven NE surges in the PFC with aging, we examined the expression levels of two proteins critical for presynaptic NE release and NE reuptake: the α2a‐adrenergic receptor and the NE transporter. Both showed a significant decrease in the PFC with aging. These findings support the concept that aging significantly alters the structural and functional dynamics within the LC‐PFC neural circuit, impacting NE modulation and neuronal activity, which may underlie the observed declines in cognitive and motor functions in aging populations. [ABSTRACT FROM AUTHOR]

Abstract: Excitation-contraction (EC) coupling in a population of skeletal muscle fibers of aged mice becomes dependent on the presence of external Ca2+ ions (Payne, A.M., Zheng, Z., Gonzalez, E., Wang, Z.M., Messi, M.L., Delbono, O., 2004b. External Ca(2+)-dependent excitation – contraction coupling in a population of aging mouse skeletal muscle fibers. J. Physiol. 560, 137–155.). However, the mechanism(s) underlying this process remain unknown. In this work, we examined the role of (1) extracellular Ca2+; (2) voltage-induced influx of external Ca2+ ions; (3) sarcoplasmic reticulum (SR) Ca2+ depletion during repeated contractions; (4) store-operated Ca2+ entry (SOCE); (5) SR ultrastructure; (6) SR subdomain localization of the ryanodine receptor; and (7) sarcolemmal excitability in muscle force decline with aging. These experiments show that external Ca2+, but not Ca2+ influx, is needed to maintain force upon repetitive fiber electrical stimulation. Decline in fiber force is associated with depressed SR Ca2+ release. SR Ca2+ depletion, SOCE, and the putative segregated Ca2+ release store do not play a significant role in external Ca2+-dependent contraction. More importantly, a significant number of action potentials fail in senescent mouse muscle fibers subjected to a stimulation frequency. These results indicate that failure to generate action potentials accounts for decreased intracellular Ca2+ mobilization and tetanic force in aging muscle exposed to a Ca2+-free medium. [Copyright &y& Elsevier]

Increasing evidence indicates that, first, the sympathetic nervous system interacts extensively with both vasculature and skeletal muscle fibers near neuromuscular junctions (NMJs) and, second, its neurotransmitter, noradrenaline, influences myofiber molecular composition and function and motor innervation. Since sympathomimetic agents have been reported to improve NMJ transmission, we examined whether two in clinical use, salbutamol and clenbuterol, affect the motor axon terminal via extracellular Ca 2+ and molecular targets, such as TRPV1 and P/Q- and N-type voltage-activated Ca 2+ channels. Electrophysiological recordings in ex-vivo preparations of peroneal nerves and lumbricalis muscles from young adult mice focused on spontaneous miniature end-plate potentials and singly and repetitively evoked end-plate potentials. Adding one dose of salbutamol or clenbuterol to the nerve/muscle preparation or repeatedly administering salbutamol to a mouse for 4 weeks increased spontaneous and evoked synaptic vesicle release but induced a steep decline in EPP amplitude in response to repetitive nerve stimulation. These effects were mediated primarily by ω-agatoxin IVA-sensitive P/Q-type and secondarily by ω-conotoxin GVIA-sensitive N-type Ca 2+ channels. Presynaptic arvanil-sensitive TRPV1 channels seem to regulate Ca 2+ at the motor neuron terminal at rest, while putative presynaptic β-adrenergic receptors may mediate sympathomimetic and catecholamine effects on presynaptic Ca 2+ channels during NMJ activation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In the present work we test the hypothesis that sustained transgenic overexpression of insulin-like growth factor-1 (IGF-1) in skeletal muscle prevents age-related decreases in myoplasmic Ca2+ concentration and consequently in specific force in single intact fibres from the flexor digitorum brevis (FDB) muscle from the mouse. Measurements of IGF-1 concentration in FDB muscle showed higher levels in transgenic than in wild-type mice at all ages. The specific tetanic force decreased significantly in single muscle fibres from old (286 +/- 22 kPa) compared to young wild-type (455 +/- 28 kPa), young transgenic (423 +/- 43 kPa), and old transgenic mice (386 +/- 15 kPa) (P < 0.05). These results are consistent with measurements in whole FDB muscles. The peak Ca2+ concentration values in response to prolonged stimulation were: 1.47 +/- 0.15, 1.70 +/- 0.29, 0.97 +/- 0.13 and 1.7 +/- 0.22 microM, in fibres from young wild-type, young transgenic, old wild-type and old transgenic mice, respectively. The effects of caffeine on FDB fibres support the conclusion that the age-related decline in peak myoplasmic Ca2+ and specific force is not explained by sarcoplasmic reticulum Ca2+ depletion. Immunohistochemistry in muscle cross-sections was performed to determine whether age and/or IGF-1 overexpression induce changes in fibre type composition. The relative percentages of type IIa, IIx and I myosin heavy chain (MHC) isoforms did not change significantly with age or genotype. Therefore, IGF-1 prevents age-related decline in peak intracellular Ca2+ and specific force in a muscle that does not exhibit changes in fibre type composition with senescence.