Showing papers in "The Journal of Physiology in 2003"

Mitochondrial formation of reactive oxygen species.

Abstract: The reduction of oxygen to water proceeds via one electron at a time. In the mitochondrial respiratory chain, Complex IV (cytochrome oxidase) retains all partially reduced intermediates until full reduction is achieved. Other redox centres in the electron transport chain, however, may leak electrons to oxygen, partially reducing this molecule to superoxide anion (O2−•). Even though O2−• is not a strong oxidant, it is a precursor of most other reactive oxygen species, and it also becomes involved in the propagation of oxidative chain reactions. Despite the presence of various antioxidant defences, the mitochondrion appears to be the main intracellular source of these oxidants. This review describes the main mitochondrial sources of reactive species and the antioxidant defences that evolved to prevent oxidative damage in all the mitochondrial compartments. We also discuss various physiological and pathological scenarios resulting from an increased steady state concentration of mitochondrial oxidants.

Pharmacological Modulation of Cortical Excitability Shifts Induced by Transcranial Direct Current Stimulation in Humans

Abstract: Transcranial direct current stimulation (tDCS) of the human motor cortex results in polarity-specific shifts of cortical excitability during and after stimulation. Anodal tDCS enhances and cathodal stimulation reduces excitability. Animal experiments have demonstrated that the effect of anodal tDCS is caused by neuronal depolarisation, while cathodal tDCS hyperpolarises cortical neurones. However, not much is known about the ion channels and receptors involved in these effects. Thus, the impact of the sodium channel blocker carbamazepine, the calcium channel blocker flunarizine and the NMDA receptor antagonist dextromethorphane on tDCS-elicited motor cortical excitability changes of healthy human subjects were tested. tDCS-protocols inducing excitability alterations (1) only during tDCS and (2) eliciting long-lasting after-effects were applied after drug administration. Carbamazepine selectively eliminated the excitability enhancement induced by anodal stimulation during and after tDCS. Flunarizine resulted in similar changes. Antagonising NMDA receptors did not alter current-generated excitability changes during a short stimulation, which elicits no after-effects, but prevented the induction of long-lasting after-effects independent of their direction. These results suggest that, like in other animals, cortical excitability shifts induced during tDCS in humans also depend on membrane polarisation, thus modulating the conductance of sodium and calcium channels. Moreover, they suggest that the after-effects may be NMDA receptor dependent. Since NMDA receptors are involved in neuroplastic changes, the results suggest a possible application of tDCS in the modulation or induction of these processes in a clinical setting. The selective elimination of tDCS-driven excitability enhancements by carbamazepine proposes a role for this drug in focussing the effects of cathodal tDCS, which may have important future clinical applications.

Exercise induces transient transcriptional activation of the PGC-1α gene in human skeletal muscle

Abstract: Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1α (PGC-1α) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell culture and rodent skeletal muscle. To determine whether PGC-1α transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl-CoA dehydrogenase mRNA, were higher in the trained than the untrained leg prior to exercise. Exercise induced a marked transient increase (P 40-fold) and mRNA content (7- to 10-fold), peaking within 2 h after exercise. Activation of PGC-1α was greater in the trained leg despite the lower relative workload. Interestingly, exercise did not affect nuclear respiratory factor 1 (NRF-1) mRNA, a gene induced by PGC-1α in cell culture. HKII, mitochondrial transcription factor A, peroxisome proliferator activated receptor α, and calcineurin Aα and Aβ mRNA were elevated (≈2- to 6-fold; P < 0.05) at 6 h of recovery in the untrained leg but did not change in the trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC-1α transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator, these findings suggest that PGC-1α may coordinate the activation of metabolic genes in human muscle in response to exercise.

A Phase Response Curve to Single Bright Light Pulses in Human Subjects

Abstract: The circadian pacemaker is differentially sensitive to the resetting effects of retinal light exposure, depending upon the circadian phase at which the light exposure occurs. Previously reported human phase response curves (PRCs) to single bright light exposures have employed small sample sizes, and were often based on relatively imprecise estimates of circadian phase and phase resetting. In the present study, 21 healthy, entrained subjects underwent pre- and post-stimulus constant routines (CRs) in dim light (approximately 2-7 lx) with maintained wakefulness in a semi-recumbent posture. The 6.7 h bright light exposure stimulus consisted of alternating 6 min fixed gaze (approximately 10 000 lx) and free gaze (approximately 5000-9000 lx) exposures. Light exposures were scheduled across the circadian cycle in different subjects so as to derive a PRC. Plasma melatonin was used to determine the phase of the onset, offset, and midpoint of the melatonin profiles during the CRs. Phase shifts were calculated as the difference in phase between the pre- and post-stimulus CRs. The resultant PRC of the midpoint of the melatonin rhythm revealed a characteristic type 1 PRC with a significant peak-to-trough amplitude of 5.02 h. Phase delays occurred when the light stimulus was centred prior to the critical phase at the core body temperature minimum, phase advances occurred when the light stimulus was centred after the critical phase, and no phase shift occurred at the critical phase. During the subjective day, no prolonged 'dead zone' of photic insensitivity was apparent. Phase shifts derived using the melatonin onsets showed larger magnitudes than those derived from the melatonin offsets. These data provide a comprehensive characterization of the human PRC under highly controlled laboratory conditions.

Pyramidal cell communication within local networks in layer 2/3 of rat neocortex

Abstract: The extent to which neocortical pyramidal cells function as a local network is determined by the strength and probability of their connections. By mapping connections between pyramidal cells we show here that in a local network of about 600 pyramidal cells located within a cylindrical volume of 200 microm x 200 microm of neocortical layer 2/3, an individual pyramidal cell receives synaptic inputs from about 30 other pyramidal neurons, with the majority of EPSP amplitudes in the 0.2-1.0 mV range. The probability of connection decreased from 0.09 to 0.01 with intercell distance (over the range 25-200 microm). Within the same volume, local interneuron (fast-spiking non-accommodating interneuron, FS)-pyramidal cell connections were about 10 times more numerous, with the majority of connections being reciprocal. The probability of excitatory and inhibitory connections between pyramidal cells and FS interneurons decreased only slightly with distance, being in the range 0.5-0.75. Pyramidal cells in the local network received strong synaptic input during stimulation of afferent fibres in layers 1 and 6. Minimal-like stimulation of layer 1 or layer 6 inputs simultaneously induced postsynaptic potentials in connected pyramidal cells as well as in pyramidal-FS cell pairs. These inputs readily induced firing of pyramidal cells, although synaptically connected cells displayed different firing patterns. Unitary EPSPs in pyramidal-pyramidal cell pairs did not detectably alter cell firing. FS interneurons fire simultaneously with pyramidal cells. In pyramidal-FS cell pairs, both unitary EPSPs and IPSPs efficiently modulated cell firing patterns. We suggest that computation in the local network may proceed not only by direct pyramidal-pyramidal cell communication but also via local interneurons. With such a high degree of connectivity with surrounding pyramidal cells, local interneurons are ideally poised to both coordinate and expand the local pyramidal cell network via pyramidal-interneuron-pyramidal communication.

Human Muscle Protein Synthesis is Modulated by Extracellular, Not Intramuscular Amino Acid Availability: A Dose‐Response Study

Abstract: To test the hypothesis that muscle protein synthesis (MPS) is regulated by the concentration of extracellular amino acids, we investigated the dose-response relationship between the rate of human MPS and the concentrations of blood and intramuscular amino acids. We increased blood mixed amino acid concentrations by up to 240 % above basal levels by infusion of mixed amino acids (Aminosyn 15, 44-261 mg kg−1 h−1) in 21 healthy subjects, (11 men 10 women, aged 29 ± 2 years) and measured the rate of incorporation of D5-phenylalanine or D3-leucine into muscle protein and blood and intramuscular amino acid concentrations. The relationship between the fold increase in MPS and blood essential amino acid concentration ([EAA], mM) was hyperbolic and fitted the equation MPS = (2.68 × [EAA])/(1.51 + [EAA]) (P < 0.01). The pattern of stimulation of myofibrillar, sarcoplasmic and mitochondrial protein was similar. There was no clear relationship between the rate of MPS and the concentration of intramuscular EAAs; indeed, when MPS was increasing most rapidly, the concentration of intramuscular EAAs was below basal levels. We conclude that the rates of synthesis of all classes of muscle proteins are acutely regulated by the blood [EAA] over their normal diurnal range, but become saturated at high concentrations. We propose that the stimulation of protein synthesis depends on the sensing of the concentration of extracellular, rather than intramuscular EAAs.

Effect of strength training on human patella tendon mechanical properties of older individuals

Abstract: This study investigated the effect of strength training on the mechanical properties of the human patella tendon of older individuals. Subjects were assigned to training (n = 9; age 74.3 +/- 3.5 years, body mass 69.7 +/- 14.8 kg and height 163.4 +/- 9.1 cm, mean +/- S.D.) and control (n = 9; age 67.1 +/- 2 years, body mass 73.5 +/- 14.9 kg and height 168.3 +/- 11.5 cm) groups. Strength training (two series of 10 repetitions at 80 % of five-repetition maximum) was performed three times per week for 14 weeks using leg extension and leg press exercises. Measurements of tendon elongation during a ramp isometric knee extension were performed before and after training and control periods in vivo using ultrasonography. Training caused a decreased tendon elongation and strain at all levels of force and stress (P 0.05). This study shows for the first time that strength training in old age increases the stiffness and Young's modulus of human tendons. This may reduce the risk of tendon injury in old age and has implications for contractile force production and the rapid execution of motor tasks.

The effect of ageing and immobilization on structure and function of human skeletal muscle fibres.

Abstract: Biopsy samples were taken from vastus lateralis muscle of seven young (YO, age 30.2 +/- 2.2 years), and seven elderly (EL, age 72.7 +/- 2.3 years) subjects and two elderly subjects whose right leg had been immobilized for 3.5 months (EL-IMM, ages 70 and 75). The following main parameters were studied: (1) myosin heavy chain (MHC) isoform distribution of the samples, determined by SDS-PAGE; (2) cross-sectional area (CSA), specific force (Po/CSA) and maximum shortening velocity (Vo) of a large population (n = 593) of single skinned muscle fibres, classified on the basis of MHC isoform composition determined by SDS-PAGE; (3) actin sliding velocity (Vf) on pure myosin isoforms determined by in vitro motility assays; (4) myosin concentration in single fibres determined by quantitative SDS-PAGE. MHC isoform distribution was shifted towards fast isoforms in EL and to a larger extent in EL-IMM. In EL and, more consistently, in EL-IMM we observed a higher percentage of hybrid fibres than in YO, and noted the presence of MHC-neonatal and of unusual hybrid fibres containing more than two MHC isoforms. Po/CSA significantly decreased in type 1 and 2A fibres in the order YO EL EL-IMM. Vo of type 1 and 2A fibres was significantly lower in EL and higher in EL-IMM than in YO, i.e. immobilization more than counteracted the age-dependent decrease in Vo. The latter phenomenon was not observed for Vf. Vf on myosin 1 was lower in both EL and EL-IMM than in YO. Vf on myosin 2X was lower in EL than in YO, and a similar trend was observed for myosin 2A. Myosin concentration decreased in type 1 and 2A fibres in the order YO EL EL-IMM and was linearly related to the Po/CSA values of corresponding fibre types from the same subjects. The experiments suggest that (1) myosin concentration is a major determinant of the lower Po/CSA of single fibres in ageing and especially following immobilization and (2) ageing is associated with lower Vo of single fibres due to changes in the properties of myosin itself, whereas immobilization is associated with higher Vo in the absence of a change in myosin function.

Suppression of skeletal muscle lipoprotein lipase activity during physical inactivity: a molecular reason to maintain daily low-intensity activity

Abstract: We have examined the regulation of lipoprotein lipase (LPL) activity in skeletal muscle during physical inactivity in comparison to low-intensity contractile activity of ambulatory controls. From studies acutely preventing ambulatory activity of one or both the hindlimbs in rats, it was shown that ≈90–95 % of the heparin-releasable (HR) LPL activity normally present in rat muscle with ambulatory activity is lost, and thus dependent on local contractile activity. Similarly, ≈95 % of the differences in LPL activity between muscles of different fibre types was dependent on ambulatory activity. The robustness of the finding that physical inactivity significantly decreases muscle LPL activity was evident from confirmatory studies with different models of inactivity, in many rats and mice, both sexes, three muscle types and during both acute and chronic (11 days) treatment. Inactivity caused a local reduction of plasma [3H]triglyceride uptake into muscle and a decrease in high density lipoprotein cholesterol concentration. LPL mRNA was not differentially expressed between ambulatory controls and either the acutely or chronically inactive groups. Instead, the process involved a rapid loss of the HR-LPL protein mass (the portion of LPL largely associated with the vascular endothelium) by an actinomycin D-sensitive signalling mechanism (i.e. transcriptionally dependent process). Significant decreases of intracellular LPL protein content lagged behind the loss of HR-LPL protein. Treadmill walking raised LPL activity ≈8-fold (P < 0.01) within 4 h after inactivity. The striking sensitivity of muscle LPL to inactivity and low-intensity contractile activity may provide one piece of the puzzle for why inactivity is a risk factor for metabolic diseases and why even non-vigorous activity provides marked protection against disorders involving poor lipid metabolism.

Depressed mitochondrial transcription factors and oxidative capacity in rat failing cardiac and skeletal muscles

Abstract: Congestive heart failure (CHF) induces alterations in energy metabolism and mitochondrial function that span cardiac as well as skeletal muscles. Whether these defects originate from altered mitochondrial DNA copy number and/or mitochondrial gene transcription is not known at present, nor are the factors that control mitochondrial capacity in different muscle types completely understood. We used an experimental model of CHF induced by aortic banding in the rat and investigated mitochondrial respiration and enzyme activity of biochemical mitochondrial markers in cardiac, slow and fast skeletal muscles. We quantified mitochondrial DNA (mtDNA), expression of nuclear (COX IV) and mitochondrial (COX I) encoded cytochrome c oxidase subunits as well as nuclear factors involved in mitochondrial biogenesis and in the necessary coordinated interplay between nuclear and mitochondrial genomes in health and CHF. CHF induced a decrease in oxidative capacity and mitochondrial enzyme activities with a parallel decrease in the mRNA level of COX I and IV, but no change in mtDNA content. The expression of the peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) gene was downregulated in CHF, as well as nuclear respiratory factor 2 and mitochondrial transcription factor A, which act downstream from PGC-1α. Most interestingly, only the level of PGC-1α expression was strongly correlated with muscle oxidative capacity in cardiac and skeletal muscles, both in healthy and CHF rats. Mitochondrial gene transcription is reduced in CHF, and PGC-1α appears as a potential modulator of muscle oxidative capacity under these experimental conditions.

Expression of IGF-I splice variants in young and old human skeletal muscle after high resistance exercise.

Abstract: The mRNA expression of two splice variants of the insulin-like growth factor-I (IGF-I) gene, IGF-IEa and mechano growth factor (MGF), were studied in human skeletal muscle. Subjects (eight young, aged 25–36 years, and seven elderly, aged 70–82 years) completed 10 sets of six repetitions of single legged knee extensor exercise at 80 % of their one repetition maximum. Muscle biopsy samples were obtained from the quadriceps muscle of both the control and exercised legs 2.5 h after completion of the exercise bout. Expression levels of the IGF-I mRNA transcripts were determined using real-time quantitative RT-PCR with specific primers. The resting levels of MGF were significantly (≈100-fold) lower than those of the IGF-IEa isoform. No difference was observed between the resting levels of the two isoforms between the two subject groups. High resistance exercise resulted in a significant increase in MGF mRNA in the young, but not in the elderly subjects. No changes in IGF-IEa mRNA levels were observed as a result of exercise in either group. The mRNA levels of the transcription factor MyoD were greater at rest in the older subjects (P < 0.05), but there was no significant effect of the exercise bout. Electrophoretic separation of myosin heavy chain (MHC) isoforms showed the older subjects to have a lower (P < 0.05) percentage of MHC-II isoforms than the young subjects. However, no association was observed between the composition of the muscle and changes in the IGF-I isoforms with exercise. The data from this study show an attenuated MGF response to high resistance exercise in the older subjects, indicative of age-related desensitivity to mechanical loading. The data in young subjects indicate that the MGF and IGF-IEa isoforms are differentially regulated in human skeletal muscle.

The human respiratory gate

Abstract: Respiratory activity phasically alters membrane potentials of preganglionic vagal and sympathetic motoneurones and continuously modulates their responsiveness to stimulatory inputs. The most obvious manifestation of this 'respiratory gating' is respiratory sinus arrhythmia, the rhythmic fluctuations of electrocardiographic R-R intervals observed in healthy resting humans. Phasic autonomic motoneurone firing, reflecting the throughput of the system, depends importantly on the intensity of stimulatory inputs, such that when levels of stimulation are low (as with high arterial pressure and sympathetic activity, or low arterial pressure and vagal activity), respiratory fluctuations of sympathetic or vagal firing are also low. The respiratory gate has a finite capacity, and high levels of stimulation override the ability of respiration to gate autonomic responsiveness. Autonomic throughput also depends importantly on other factors, including especially, the frequency of breathing, the rate at which the gate opens and closes. Respiratory sinus arrhythmia is small at rapid, and large at slow breathing rates. The strong correlation between systolic pressure and R-R intervals at respiratory frequencies reflects the influence of respiration on these two measures, rather than arterial baroreflex physiology. A wide range of evidence suggests that respiratory activity gates the timing of autonomic motoneurone firing, but does not influence its tonic level. I propose that the most enduring significance of respiratory gating is its use as a precisely controlled experimental tool to tease out and better understand otherwise inaccessible human autonomic neurophysiological mechanisms.

The rate of protein digestion affects protein gain differently during aging in humans

Abstract: In young men ingesting protein meals, slowly digested proteins (caseins: CAS) induce a higher protein gain than those that are rapidly digested (whey proteins: WP). Our aim was to assess whether or not this is true in elderly men receiving mixed meals. The effects of meals containing either CAS or two different amounts of WP (WP-iN: isonitrogenous with CAS, or WP-iL: providing the same amount of leucine as CAS) on protein metabolism (assessed by combining oral and intravenous leucine tracers) were compared in nine healthy, elderly (mean +/- S.E.M. age 72 +/- 1 years) and six young men (24 +/- 1 years). In both age groups, WP-iL and WP-iN were digested faster than CAS (P < 0.001, ANOVA). Proteolysis was inhibited similarly whatever the meal and age groups (P = NS). Protein synthesis was higher with WP-iN than with CAS or WP-iL (P < 0.01), irrespective of age (P = NS). An age-related effect (P < 0.05) was found with postprandial leucine balance. Leucine balance was higher with CAS than with WP-iL (P < 0.01) in young men, but not in elderly subjects (P = NS). In isonitrogenous conditions, leucine balance was higher with WP-iN than with CAS (P < 0.001) in both age groups, but the magnitude of the differences was higher in the elderly men (P = 0.05). In conclusion, during aging, protein gain was greater with WP (rapidly digested protein), and lower with CAS (slowly digested protein). This suggests that a 'fast' protein might be more beneficial than a 'slow' one to limit protein losses during aging.

Measurement of voluntary activation of fresh and fatigued human muscles using transcranial magnetic stimulation.

Abstract: Recently, transcranial magnetic stimulation of the motor cortex (TMS) revealed impaired voluntary activation of muscles during maximal efforts. Hence, we evaluated its use as a measure of voluntary activation over a range of contraction strengths in both fresh and fatigued muscles, and compared it with standard twitch interpolation using nerve stimulation. Subjects contracted the elbow flexors isometrically while force and EMG from biceps and triceps were recorded. In one study, eight subjects made submaximal and maximal test contractions with rests to minimise fatigue. In the second study, eight subjects made sustained maximal contractions to reduce force to 60 % of the initial value, followed by brief test contractions. Force responses were recorded following TMS or electrical stimulation of the biceps motor nerve. In other contractions, EMG responses to TMS (motor evoked potentials, MEPs) or to stimulation at the brachial plexus (maximal M waves, Mmax) were recorded. During contractions of 50 % maximum, TMS elicited large MEPs in biceps (> 90 % Mmax) which decreased in size (to approximately 70 % Mmax) with maximal efforts. This suggests that faster firing rates made some motor units effectively refractory. With fatigue, MEPs were also smaller but remained > 70 % Mmax for contractions of 50-100 % maximum. For fresh and fatigued muscle, the superimposed twitch evoked by motor nerve and motor cortex stimulation decreased with increasing contraction strength. For nerve stimulation the relation was curvilinear, and for TMS it was linear for contractions of 50-100 % maximum (r2 = 1.00). Voluntary activation was derived using the expression: (1 - superimposed twitch/resting twitch) x 100. The resting twitch was measured directly for nerve stimulation and for TMS, it was estimated by extrapolation of the linear regression between the twitch and voluntary force. For cortical stimulation, this resulted in a highly linear relation between voluntary activation and force. Furthermore, the estimated activation corresponded well with contraction strength. Using TMS or nerve stimulation, voluntary activation was high during maximal efforts of fresh muscle. With fatigue, both measures revealed reduced voluntary activation (i.e. central fatigue) during maximal efforts. Measured with TMS, this central fatigue accounted for one-quarter of the fall in maximal voluntary force. We conclude that TMS can quantify voluntary activation for fresh or fatigued muscles at forces of 50-100 % maximum. Unlike standard twitch interpolation of the elbow flexors, voluntary activation measured with TMS varies in proportion to voluntary force, it reveals when extra output is available from the motor cortex to increase force, and it elicits force from all relevant synergist muscles.

Intermittent hypoxia increases insulin resistance in genetically obese mice.

Abstract: Obstructive sleep apnoea, a syndrome that leads to recurrent intermittent hypoxia, is associated with insulin resistance in obese individuals, but the mechanisms underlying this association remain unknown. We utilized a mouse model to examine the effects of intermittent hypoxia on insulin resistance in lean C57BL/6J mice and leptin-deficient obese (C57BL/6J−Lepob) mice. In lean mice, exposure to intermittent hypoxia for 5 days (short term) resulted in a decrease in fasting blood glucose levels (from 173 ± 11 mg dl−1 on day 0 to 138 ± 10 mg dl−1 on day 5, P < 0.01), improvement in glucose tolerance without a change in serum insulin levels and an increase in serum leptin levels in comparison with control (2.6 ± 0.3 vs. 1.7 ± 0.2 ng ml−1, P < 0.05). Microarray mRNA analysis of adipose tissue revealed that leptin was the only upregulated gene affecting glucose uptake. In obese mice, short-term intermittent hypoxia led to a decrease in blood glucose levels accompanied by a 607 ± 136 % (P < 0.01) increase in serum insulin levels. This increase in insulin secretion after 5 days of intermittent hypoxia was completely abolished by prior leptin infusion. Obese mice exposed to intermittent hypoxia for 12 weeks (long term) developed a time-dependent increase in fasting serum insulin levels (from 3.6 ± 1.1 ng ml−1 at baseline to 9.8 ± 1.8 ng ml−1 at week 12, P < 0.001) and worsening glucose tolerance, consistent with an increase in insulin resistance. We conclude that the increase in insulin resistance in response to intermittent hypoxia is dependent on the disruption of leptin pathways.

Regulation of supply and demand for maternal nutrients in mammals by imprinted genes

Abstract: The placenta has evolved in eutherian mammals primarily to provide nutrients for the developing fetus. The genetic control of the regulation of supply and demand for maternal nutrients is not understood. In this review we argue that imprinted genes have central roles in controlling both the fetal demand for, and the placental supply of, maternal nutrients. Recent studies on Igf2 (insulin-like growth factor 2) knockout mouse models provide experimental support for this hypothesis. These show effects on placental transport capacity consistent with a role of IGF-II in modulating both the placental supply and fetal demand for nutrients. Imprinting of genes with such functions may have coevolved with the placenta and new evidence suggests that transporter proteins, as well as the regulators themselves, may also be imprinted. These data and hypotheses are important, as deregulation of supply and demand affects fetal growth and has long term consequences for health in mammals both in the neonatal period and, as a result of fetal programming, in adulthood.

Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart

Abstract: Opening of the mitochondrial permeability transition pore (MPTP) is thought to be a critical event in mediating the damage to hearts that accompanies their reperfusion following prolonged ischaemia Protection from reperfusion injury occurs if the prolonged ischaemic period is preceded by short ischaemic periods followed by recovery Here we investigate whether such ischaemic preconditioning (IPC) is accompanied by inhibition of MPTP opening MPTP opening in Langendorff-perfused rat hearts was determined by perfusion with 2-deoxy[3H]glucose ([3H]DOG) and measurement of mitochondrial [3H]DOG entrapment We demonstrate that IPC inhibits initial MPTP opening in hearts reperfused after 30 min global ischaemia, and subsequently enhances pore closure as hearts recover However, MPTP opening in mitochondria isolated from IPC hearts occurred more readily than control mitochondria, implying that MPTP inhibition by IPC in situ was secondary to other factors such as decreased calcium overload and oxidative stress Hearts perfused with cyclosporin A or sanglifehrin A, powerful inhibitors of the MPTP, also recovered better from ischaemia than controls (improved haemodynamic function and less lactate dehydrogenase release) However, the mitochondrial DOG entrapment technique showed these agents to be less effective than IPC at preventing MPTP opening Our data suggest that protection from reperfusion injury is better achieved by reducing factors that induce MPTP opening than by inhibiting the MPTP directly

Single Muscle Fibre Contractile Properties in Young and Old Men and Women

Abstract: The purpose of this study was to determine whether there was an age-related decline in the isometric and isotonic contractile function of permeabilized slow (MHC I) and fast (MHC IIa) single muscle fibres. Vastus lateralis muscle fibres from six young men (YM; 25 +/- 1 years), six young women (YW; 25 +/- 1 years), six old men (OM; 80 +/- 4 years) and six old women (OW; 78 +/- 2 years) were studied at 15 degrees C for in vitro force-velocity properties, peak force and contractile velocity. Peak power was 23-28 % lower (P 0.05) among the four groups. These data suggest that differences in whole muscle strength and function that are often observed with ageing appear to be regulated by quantitative rather than qualitative parameters of single muscle fibre contractile function.

The TTX‐Resistant Sodium Channel Nav1.8 (SNS/PN3): Expression and Correlation with Membrane Properties in Rat Nociceptive Primary Afferent Neurons

Abstract: We have examined the distribution of the sensory neuron-specific Na+ channel Nav1.8 (SNS/PN3) in nociceptive and non-nociceptive dorsal root ganglion (DRG) neurons and whether its distribution is related to neuronal membrane properties. Nav1.8-like immunoreactivity (Nav1.8-LI) was examined with an affinity purified polyclonal antiserum (SNS11) in rat DRG neurons that were classified according to sensory receptive properties and by conduction velocity (CV) as C-, Aδ- or Aα/β. A significantly higher proportion of nociceptive than low threshold mechanoreceptive (LTM) neurons showed Nav1.8-LI, and nociceptive neurons had significantly more intense immunoreactivity in their somata than LTM neurons. Results showed that 89, 93 and 60 % of C-, Aδ- and Aα/β-fibre nociceptive units respectively and 88 % of C-unresponsive units were positive. C-unresponsive units had electrical membrane properties similar to C-nociceptors and were considered to be nociceptive-type neurons. Weak positive Nav1.8-LI was also present in some LTM units including a C LTM, all Aδ LTM units (D hair), about 10 % of cutaneous LTM Aα/β-units, but no muscle spindle afferent units. Nav1.8-LI intensity was negatively correlated with soma size (all neurons) and with dorsal root CVs in A- but not C-fibre neurons. Nav1.8-LI intensity was positively correlated with action potential (AP) duration (both rise and fall time) in A-fibre neurons and with AP rise time only in positive C-fibre neurons. It was also positively correlated with AP overshoot in positive neurons. Thus high levels of Nav1.8 protein may contribute to the longer AP durations (especially in A-fibre neurons) and larger AP overshoots that are typical of nociceptors.

Ghrelin Induces Fasted Motor Activity of the Gastrointestinal Tract in Conscious Fed Rats

Abstract: Ghrelin is a newly discovered orexigenic peptide originating from the stomach. However, its action in regulating the fed and fasted motor activity of the digestive tract is not fully understood. In the present study, we examined the effects of intracerebroventricular (i.c.v.) and intravenous (i.v.) injection of ghrelin on the physiological fed and fasted motor activities in the stomach and duodenum of freely moving conscious rats. i.c.v. and i.v. injection of ghrelin induced fasted motor activity in the duodenum in normal fed rats, while i.v. injection of ghrelin induced fasted motor activity in both the stomach and duodenum in vagotomized rats. The effects of i.c.v. and i.v. injected ghrelin were blocked by growth hormone secretagogue receptor (GHS-R) antagonist given by the same route and also blocked by immunoneutralization of neuropeptide Y (NPY) in the brain. The effects of i.v. injected ghrelin were not altered by i.c.v. injection of GHS-R antagonist in vagotomized rats. Injection of GHS-R antagonist blocked the fasted motor activity in both the stomach and duodenum in vagotomized rats but did not affect the fasted motor activity in normal rats. Low intragastric pH inhibited the effect of ghrelin. The present results indicate that ghrelin is involved in regulation of fasted motor activity in the stomach and duodenum. Peripheral ghrelin may induce the fasted motor activity by activating the NPY neurons in the brain, probably through ghrelin receptors on vagal afferent neurons. Once the brain mechanism is eliminated by truncal vagotomy, ghrelin might be primarily involved in the regulation of fasted motor activity through ghrelin receptors on the stomach and duodenum. The action of ghrelin to induce fasted motor activity is strongly affected by intragastric pH; low pH inhibits the action.

Signalling pathways involved in the sensitisation of mouse nociceptive neurones by nerve growth factor.

Abstract: Nerve growth factor (NGF) causes a rapid sensitisation of nociceptive sensory neurones to painful thermal stimuli owing to an action on the heat and capsaicin receptor TRPV1 (formerly known as VR1). We have developed a new technique to study this rapid sensitisation of TRPV1 by monitoring the effects of NGF on the increase in intracellular calcium concentration ([Ca2+]i) following exposure to capsaicin. Brief applications of capsaicin caused a rise in [Ca2+]i, and NGF was found to enhance this rise in 37 % of capsaicin-responsive neurones within 2 min. Pathways responsible for transducing the sensitisation of TRPV1 by TrkA, the NGF receptor, were characterised by observing the effects of inhibitors of key members of NGF-activated second messenger signalling cascades. Specific inhibitors of the ras/MEK (mitogen-activated protein and extracellular signal-regulated kinases) pathway and of phospholipase C did not abolish the NGF-induced sensitisation, but wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K), totally abolished the effect of NGF. Pharmacological blockade of protein kinase C (PKC) or calcium-calmodulin-dependent protein kinase II (CaMK II) activation also prevented NGF-induced sensitisation, while blockade of protein kinase A (PKA) was without effect. These data indicate that the crucial early pathway activated by NGF involves PI3K, while PKC and CaMK II are also involved, probably at subsequent stages of the NGF-activated signalling pathway.

Expression and splicing of the insulin-like growth factor gene in rodent muscle is associated with muscle satellite (stem) cell activation following local tissue damage.

Abstract: Muscle satellite cells are mononuclear cells that remain in a quiescent state until activated when they proliferate and fuse with muscle fibres to donate nuclei, a process necessary for post-embryonic growth, hypertrophy and tissue repair in this post-mitotic tissue. These processes have been associated with expression of the insulin-like growth factor (IGF-I) gene that can undergo alternative splicing to generate different gene products with varying functions. To gain insight into the cellular mechanisms involved in local tissue repair, the time courses of expression of two IGF-I splice variants produced in muscle were determined together with marker genes for satellite cell activation following local muscle damage. Using real-time RT-PCR with specific primers, the mRNA transcripts in rat tibialis anterior muscles were measured at different time intervals following either mechanical damage imposed by electrical stimulation of the stretched muscle or damage caused by injection with bupivacaine. It was found that the autocrine splice variant mechano growth factor (MGF) was rapidly expressed and then declined within a few days following both types of damage. Systemic IGF-IEa was more slowly upregulated and its increase was commensurate with the rate of decline in MGF expression. Satellite cell activation as measured by M-cadherin and one of the muscle regulatory factors MyoD and the sequence of expression suggests that the initial pulse of MGF is responsible for satellite cell activation, as the systemic IGF-IEa mRNA expression peaks after the expression of these markers, including M-cadherin protein. Later splicing of the IGF-I gene away from MGF but towards IGF-IEa seems physiologically appropriate as IGF-IEa is the main source of mature IGF-I for upregulation of protein synthesis required to complete the repair.

Sympathetic neural overactivity in healthy humans after prolonged exposure to hypobaric hypoxia.

Abstract: Acute exposure to hypoxia causes chemoreflex activation of the sympathetic nervous system. During acclimatization to high altitude hypoxia, arterial oxygen content recovers, but it is unknown to what degree sympathetic activation is maintained or normalized during prolonged exposure to hypoxia. We therefore measured sympathetic nerve activity directly by peroneal microneurography in eight healthy volunteers (24 ± 2 years of age) after 4 weeks at an altitude of 5260 m (Chacaltaya, Bolivian Andes) and at sea level (Copenhagen). The subjects acclimatized well to altitude, but in every subject sympathetic nerve activity was highly elevated at altitude vs. sea level (48 ± 5 vs. 16 ± 3 bursts min−1, respectively, P < 0.05), coinciding with increased mean arterial blood pressure (87 ± 3 vs. 77 ± 2 mmHg, respectively, P < 0.05). To examine the underlying mechanisms, we administered oxygen (to eliminate chemoreflex activation) and saline (to reduce cardiopulmonary baroreflex deactivation). These interventions had minor effects on sympathetic activity (48 ± 5 vs. 38 ± 4 bursts min−1, control vs. oxygen + saline, respectively, P < 0.05). Moreover, sympathetic activity was still markedly elevated (37 ± 5 bursts min−1) when subjects were re-studied under normobaric, normoxic and hypervolaemic conditions 3 days after return to sea level. In conclusion, acclimatization to high altitude hypoxia is accompanied by a striking and long-lasting sympathetic overactivity. Surprisingly, chemoreflex activation by hypoxia and baroreflex deactivation by dehydration together could account for only a small part of this response, leaving the major underlying mechanisms unexplained.

Distinct repriming and closed-state inactivation kinetics of Nav1.6 and Nav1.7 sodium channels in mouse spinal sensory neurons.

Abstract: While large, myelinated dorsal root ganglion (DRG) neurons are capable of firing at high frequencies, small unmyelinated DRG neurons typically display much lower maximum firing frequencies. However, the molecular basis for this difference has not been delineated. Because the sodium currents in large DRG neurons exhibit rapid repriming (recovery from inactivation) kinetics and the sodium currents in small DRG neurons exhibit predominantly slow repriming kinetics, it has been proposed that differences in sodium channels might contribute to the determination of repetitive firing properties in DRG neurons. A recent study demonstrated that Nav1.7 expression is negatively correlated with conduction velocity and DRG cell size, while the Nav1.6 voltage-gated sodium channel has been implicated as the predominant isoform present at nodes of Ranvier of myelinated fibres. Therefore we characterized and compared the functional properties, including repriming, of recombinant Nav1.6 and Nav1.7 channels expressed in mouse DRG neurons. Both Nav1.6 and Nav1.7 channels generated fast-activating and fast-inactivating currents. However recovery from inactivation was significantly faster (approximately 5-fold at -70 mV) for Nav1.6 currents than for Nav1.7 currents. The recovery from inactivation of Nav1.6 channels was also much faster than that of native tetrodotoxin-sensitive sodium currents recorded from small spinal sensory neurons, but similar to that of tetrodotoxin-sensitive sodium currents recorded from large spinal sensory neurons. Development of closed-state inactivation was also much faster for Nav1.6 currents than for Nav1.7 currents. Our results indicate that the firing properties of DRG neurons can be tuned by regulating expression of different sodium channel isoforms that have distinct repriming and closed-state inactivation kinetics.

Cellular and Network Mechanisms Underlying Spontaneous Sharp Wave–Ripple Complexes in Mouse Hippocampal Slices

Abstract: The mammalian hippocampus displays a peculiar pattern of fast (approximately 200 Hz) network oscillations superimposed on slower sharp waves. Such sharp wave-ripple complexes (SPW-R) have been implicated in memory consolidation. We have recently described a novel and unique method for studying SPW-R in naive slices of murine hippocampus. Here, we used this model to analyse network and cellular mechanisms of this type of network activity. SPW-R are usually generated within area CA3 but can also originate within the isolated CA1 region. Cellular synchronisation during SPW-R requires both excitatory and inhibitory synaptic transmission as well as electrical coupling, the latter being particularly important for the high-frequency component. Extracellular and intracellular recordings revealed a surprisingly strong inhibition of most CA1 pyramidal cells during SPW-R. A minority of active cells, however, increases action potential frequency and fires in strict synchrony with the field ripples. This strong separation between members and non-members of the network may serve to ensure a high signal-to-noise ratio in information processing during sharp wave-ripple complexes.

Mitogen-activated protein kinase (MAPK) pathway activation: effects of age and acute exercise on human skeletal muscle.

Abstract: The purpose of this investigation was to examine the activation (phosphorylation) and total protein content of MAPK signalling cascade proteins (ERK 1/2, p90RSK, Mnk 1, eIF4E, p38 MAPK, JNK/SAPK, MKP 1) at rest and following exercise, in sedentary young and old men. Eight young (22 ± 1 years; YM) and eight old (79 ± 3 years; OM) men underwent a resting muscle biopsy of the vastus lateralis; they then performed a knee extensor resistance exercise session (29 contractions at ∼70 % of max), followed by a post-exercise biopsy. Western immunoblot analysis demonstrated that the OM had higher resting phosphorylation of ERK 1/2, p90RSK, Mnk 1, p38 MAPK and JNK/SAPK proteins versus YM (P < 0.05). The resistance exercise bout caused an increase in phosphorylation of the ERK 1/2, p90RSK and Mnk 1 proteins (P < 0.05) in the YM. Conversely, the OM had a decrease in ERK 1/2, p90RSK, Mnk 1, p38 MAPK and JNK/SAPK phosphorylation (P < 0.05) after the exercise bout. Neither group showed a change in eIF4E phosphorylation. The total amount of protein expression of the MAPK signalling proteins was not different between the YM and OM, except that there was a higher (P < 0.05) MKP 1 protein content in the OM. This investigation is the first to provide evidence that MAPK proteins are differentially activated at rest and in response to a bout of resistance exercise in skeletal muscle of young and old men. These findings may have implications for other processes (e.g. transcription and translation) involved in skeletal muscle type and growth, when examining the changes occurring with ageing muscle before and after resistance exercise/training.

Development of rat CA1 neurones in acute versus organotypic slices: role of experience in synaptic morphology and activity.

Abstract: Despite their wide use, the physiological relevance of organotypic slices remains controversial. Such cultures are prepared at 5 days postnatal. Although some local circuitry remains intact, they develop subsequently in isolation from the animal and hence without plasticity due to experience. Development of synaptic connectivity and morphology might be expected to proceed differently under these conditions than in a behaving animal. To address these questions, patch-clamp techniques and confocal microscopy were used in the CA1 region of the rat hippocampus to compare acute slices from the third postnatal week with various stages of organotypic slices. Acute slices prepared at postnatal days (P) 14, 17 and 21 were found to be developmentally equivalent to organotypic slices cultured for 1, 2 and 3 weeks, respectively, in terms of development of synaptic transmission and dendritic morphology. The frequency of inhibitory and excitatory miniature synaptic currents increased in parallel. Development of dendritic length and primary branching as well as spine density and proportions of different spine types were also similar in both preparations, at these corresponding stages. The most notable difference between organotypic and acute slices was a four- to five-fold increase in the absolute frequency of glutamatergic (but not GABAergic) miniature postsynaptic currents in organotypic slices. This was probably related to an increase in complexity of higher order dendritic branching in organotypic slices, as measured by fractal analysis, resulting in an increased total synapse number. Both increased excitatory miniature synaptic current frequency and dendritic complexity were already established during the first week in culture. The level of complexity then stayed constant in both preparations over subsequent stages, with synaptic frequency increasing in parallel. Thus, although connectivity was greater in organotypic slices, once this was established, development continued in both preparations at a remarkably similar rate. We conclude that, for the parameters studied, changes seem to be preprogrammed by 5 days and their subsequent development is largely independent of environment.

Dynamic Receptive Fields of Reconstructed Pyramidal Cells in Layers 3 and 2 of Rat Somatosensory Barrel Cortex

Abstract: A major aim of sensory physiology is to identify those synaptic connections in cortical representational areas (functional maps) by which sensory stimuli are transformed into a specific pattern of sub- (PSPs) and suprathreshold (APs) electrical activity. In the neocortex such maps consist of functional units, referred to as columns (Mountcastle, 1957; Hubel & Wiesel, 1962). These comprise the cells in different cortical layers that respond to a particular sensory stimulus. To understand sensory maps mechanistically and at a subcellular resolution, firstly the synaptic connections between cells that constitute a column and also those between different columns have to be identified in a layer-specific manner. Secondly the spatial and temporal transformations of PSP and AP patterns along sensory pathways and in the different cortical layers have to be understood. The coarse layout of sensory information flow within a column is comparable across different sensory cortices. Afferent signals arrive in cortical layer 4 (L4) from thalamic nuclei. They are relayed from L4 to supragranular layers 3 (L3) and 2 (L2) as well as to infragranular layers (L5 and L6). Extracellular unit recording and anatomical work have compiled a detailed picture of the columnar cytoarchitecture and AP activity in columns of some sensory cortices. The detailed anatomy and synaptic mechanisms of the connections that generate specific patterns of PSPs and APs are, however, largely unclear. Few studies have determined both the soma location and the dendritic and axonal morphology of cortical cells as well as their sub- and suprathreshold RFs (e.g. Ito, 1992; Brecht & Sakmann, 2002a,b). Such measurements are, however, a prerequisite if one wants to infer how PSPs or APs represent a sensory stimulus in the different layers of the cortex. L4 of the rodent somatosensory cortex contains aggregates of neuronal somata referred to as barrels, which are innervated in a strict topographical order by inputs representing individual facial whiskers (Woolsey & Van der Loos, 1970). Anatomical studies have demonstrated that barrel cells are targeted by thalamic inputs from the ventral posterior medial nucleus (VPM), which are part of the lemniscal pathway (Diamond, 1995), while the septa between barrels are innervated by thalamic afferents projecting from the posterior medial nucleus (POM), which belong to the paralemniscal pathway (Koralek et al. 1988; Lu & Lin, 1993). While most lemniscal afferents innervate the barrels, some VPM inputs also target the L5B/L6 border and paralemniscal POM afferents densely innervate L5A (Koralek et al. 1988; Lu & Lin, 1993). Barrel borders and the morphology of a cortical cell can be visualised simultaneously (Ito, 1992), such that the laminar position of a cell and its position relative to barrel column borders as well as its detailed dendritic and axonal morphology can be measured. Such techniques provided physiological evidence that lemniscal (the VPM/barrel projection) and paralemniscal (the POM/septum projection) pathways are largely segregated in L4 (Brecht & Sakmann, 2002a). Furthermore the RFs of barrel and septum cells are dynamic but are narrow and restricted to a PW and at most the first order SuWs. The homogeneous appearance of L3 and L2 in the horizontal plane may indicate merging of the whisker-specific anatomical pathways, whose strict separation in L4 gives rise to the discontinuous appearance of barrels (Woolsey & Van der Loos, 1970). The projection pattern of L4 spiny neuron axons suggests, however, that selectively connected barrel columns also exist (Petersen & Sakmann, 2000; Petersen et al. 2003; Lubke et al. 2003). The convergence of whisker-evoked responses between columns is also suggested by unit recordings from unidentified cells (Simons, 1978, 1995; Armstrong-James & Fox, 1987; Armstrong-James et al. 1992; Armstrong-James, 1995). They show that suprathreshold RFs in L3 and L2 cells are larger in size than those of L4 cells. The work of Ahissar and colleagues on the representation of temporal frequencies in L2/3 cell spike trains suggests a merging of barrel and septum inputs in supragranular layers (Ahissar et al. 2001). Anatomical data, however, suggest that barrel and septal pathways also remain separate in L3 and L2 (Kim & Ebner, 1999). We report here in vivo whole-cell voltage recordings of whisker-evoked PSPs and APs from cells in L2/3, combined with reconstruction of their dendritic and axonal arbors. We determined the horizontal and vertical position of these cells with reference to the barrel map to establish relationships between individual cell classes in L3 and L2 located above barrel and septa, and their sub- and suprathreshold RFs. The aim is to construct relationships between anatomical cell classes and their functional properties. Comparison with similar data from L4 cells (Brecht & Sakmann, 2002b) and incorporation of in vitro data (Feldmeyer et al. 2002; Lubke et al. 2003) on connectivity should allow a quantitative description of the flow of excitation through and between cortical barrel columns.

PSD-95 regulates synaptic transmission and plasticity in rat cerebral cortex.

Abstract: PSD-95 is one of the most abundant proteins found in the postsynaptic density of excitatory synapses However, the precise functional role played by PSD-95 in regulating synaptic transmission and plasticity remains undefined To address this issue, we have overexpressed PSD-95 in cortical pyramidal neurons in organotypic brain slices using particle-mediated gene transfer and assessed the consequences on synaptic transmission and plasticity The AMPA receptor/NMDA receptor (AMPAR/NMDAR) ratio of evoked EPSCs recorded at +40 mV was greater in PSD-95-transfected pyramidal neurons than in controls This difference could not be accounted for by a change in rectification of AMPAR-mediated synaptic currents since the current-voltage curves obtained in controls and in PSD-95-transfected neurons were indistinguishable However, the amplitude of AMPAR-mediated evoked EPSCs was larger in PSD-95-transfected neurons compared to matched controls Paired-pulse ratio analysis suggested that overexpression of PSD-95 did not alter presynaptic release probability Transfection of PSD-95 was further accompanied by an increase in the frequency, but not amplitude, of AMPAR-mediated mEPSCs Together, these results indicate that transfection of PSD-95 increased AMPAR-mediated synaptic transmission Furthermore, they suggest that this phenomenon reflects an increased number of synapses expressing AMPARs rather than an increased number or function of these receptors at individual synapses We tested the consequences of these changes on synaptic plasticity and found that PSD-95 transfection greatly enhanced the probability of observing long-term depression These results thus identify a physiological role for PSD-95 and demonstrate that this protein can play a decisive role in controlling synaptic strength and activity-dependent synaptic plasticity

Effect of Vitamin C Supplements on Antioxidant Defence and Stress Proteins in Human Lymphocytes and Skeletal Muscle

Abstract: Oxidative stress induces adaptations in the expression of protective enzymes and heat shock proteins (HSPs) in a variety of tissues. We have examined the possibility that supplementation of subjects with the nutritional antioxidant, vitamin C, influences the ability of lymphocytes to express protective enzymes and HSPs following exposure to an exogenous oxidant and the response of skeletal muscle to the physiological oxidative stress that occurs during exercise in vivo. Our hypothesis was that an elevation of tissue vitamin C content would reduce oxidant-induced expression of protective enzymes and HSP content. Lymphocytes from non-supplemented subjects responded to hydrogen peroxide with increased activity of superoxide dismutase (SOD) and catalase, and HSP60 and HSP70 content over 48 h. Vitamin C supplementation at a dose of 500 mg day−1 for 8 weeks was found to increase the serum vitamin C concentration by ∼50 %. Lymphocytes from vitamin C-supplemented subjects had increased baseline SOD and catalase activities and an elevated HSP60 content. The SOD and catalase activities and the HSP60 and HSP70 content of lymphocytes from supplemented subjects did not increase significantly in response to hydrogen peroxide. In non-supplemented subjects, a single period of cycle ergometry was found to significantly increase the HSP70 content of the vastus lateralis. Following vitamin C supplementation, the HSP70 content of the muscle was increased at baseline with no further increase following exercise. We conclude that, in vitamin C-supplemented subjects, adaptive responses to oxidants are attenuated, but that this may reflect an increased baseline expression of potential protective systems against oxidative stress (SOD, catalase and HSPs).