Autism-like behavioral phenotypes in BTBR T+tf/J mice.

Autism is a behaviorally defined neurodevelopmental disorder of unknown etiology. Mouse models with face validity to the core symptoms offer an experimental approach to test hypotheses about the causes of autism and translational tools to evaluate potential treatments. We discovered that the inbred mouse strain BTBR T+tf/J (BTBR) incorporates multiple behavioral phenotypes relevant to all three diagnostic symptoms of autism. BTBR displayed selectively reduced social approach, low reciprocal social interactions and impaired juvenile play, as compared with C57BL/6J (B6) controls. Impaired social transmission of food preference in BTBR suggests communication deficits. Repetitive behaviors appeared as high levels of self-grooming by juvenile and adult BTBR mice. Comprehensive analyses of procedural abilities confirmed that social recognition and olfactory abilities were normal in BTBR, with no evidence for high anxiety-like traits or motor impairments, supporting an interpretation of highly specific social deficits. Database comparisons between BTBR and B6 on 124 putative autism candidate genes showed several interesting single nucleotide polymorphisms (SNPs) in the BTBR genetic background, including a nonsynonymous coding region polymorphism in Kmo. The Kmo gene encodes kynurenine 3-hydroxylase, an enzyme-regulating metabolism of kynurenic acid, a glutamate antagonist with neuroprotective actions. Sequencing confirmed this coding SNP in Kmo, supporting further investigation into the contribution of this polymorphism to autism-like behavioral phenotypes. Robust and selective social deficits, repetitive self-grooming, genetic stability and commercial availability of the BTBR inbred strain encourage its use as a research tool to search for background genes relevant to the etiology of autism, and to explore therapeutics to treat the core symptoms.

Estimation of body composition in muscular dystrophy by MRI and stereology.

We have applied the Cavalieri method of modern design stereology with magnetic resonance imaging for estimating the volume of whole-body muscle and fat compartments in four patients with muscular dystrophy, a patient with myopathy, five controls, an anorexic subject, and a body builder. Detailed systematic series (ie, 50) of axial MR images (T1-weighted, TR/TE 400/10 msec) were obtained throughout the whole body of each subject. The results showed that 15, 20, and 35 axial sections through the body are sufficient to secure coefficients of error (CEs) on the estimates of total muscle and fat volume of around 10%, 5%, and 3% respectively in muscular dystrophy patients and controls. The mean normalized volumes of muscle in four muscular dystrophy patients were decreased by 27% (t-test: P < 0.05), and those of total fat were increased by 12% (t-test: P > 0.05) relative to controls. The Cavalieri method provides a direct, efficient, and mathematically unbiased approach for studying human body compartments and may have application in assessing treatment efficacy in patients with muscular dystrophy. J. Magn. Reson. Imaging 2000;12:467-475.

The effect of motor learning in facioscapulohumeral muscular dystrophy patients.

Two models of motor learning (ML) practice (complex and simple) were used to investigate upper limb function in individuals with facioscapulohumeral dystrophy (FSHD). The effect of ML practice was studied by examining changes in the ability to undertake a simulated drinking task. In the complex model, seven FSHD patients and seven age- and gender-matched healthy volunteers were studied. Performance was assessed by measurement of the electromyographic (EMG) activity of the biceps brachii and brachioradialis, elbow joint flexion, shoulder joint flexion and abduction, maximum acceleration at the onset of movement, movement time and reaction time, both before and after a 30-min complex ML task. In the simple model, a second group of six FSHD patients and six age- and gender-matched healthy subjects were studied. The same parameters were measured as for the complex ML task, except that the EMG activity of the triceps and deltoid muscles (anterior part) were measured instead of that of the brachioradialis. In both studies, the FSHD patients showed significantly larger values for all parameters except the flexion of shoulder joint and reaction time, compared with controls before the ML task. In the FSHD group, while the complex ML resulted in decreases in the brachioradialis EMG activity (P<0.005) and reaction time (P<0.0001), the simple ML model resulted in significant changes towards the normal value in all parameters measured except shoulder flexion. The change in the measured variables towards normal values indicates that ML may help to improve performance in FSHD.

The role of tryptophan in fatigue in different conditions of stress.

Tryptophan is the precursor for the neurotransmitter 5-hydroxytryptamine (5-HT), which is involved in fatigue and sleep. It is present in bound and free from in the blood, where the concentration is controlled by albumin binding to tryptophan. An increase in plasma free tryptophan leads to an increased rate of entry of tryptophan into the brain. This should lead to a higher level of 5-HT which may cause central fatigue. Central fatigue is implicated in clinical conditions such as chronic fatigue syndrome and post-operative fatigue. Increased plasma free tryptophan leads to an increase in the plasma concentration ratio of free tryptophan to the branched chain amino acids (BCAA) which compete with tryptophan for entry into the brain across the blood-brain barrier. The plasma concentrations of these amino acids were measured in chronic fatigue syndrome patients (CFS) before and after exercise (Castell et al., 1998), and in patients undergoing major surgery (Yamamoto et al., 1997). In the CFS patients, the pre-exercise concentration of plasma free tryptophan was higher than in controls (p < 0.05) but did not change during or after exercise. This might indicate an abnormally high level of brain 5-HT in CFS patients leading to persistent fatigue. In the control group, plasma free tryptophan was increased after maximal exercise (p < 0.001), returning towards baseline levels 60 min later. The apparent failure of the CFS patients to change the plasma free tryptophan concentration or the free tryptophan/BCAA ratio during exercise may indicate increased sensitivity of brain 5-HT receptors, as has been demonstrated in other studies (Cleare et al., 1995). In post-operative recovery after major surgery plasma free tryptophan concentrations were markedly increased compared with baseline levels; the plasma free tryptophan/BCAA concentration ratio was also increased after surgery. Plasma albumin concentrations were decreased after surgery: this may account for the increase in plasma free tryptophan levels. Provision of BCAA has improved mental performance in athletes after endurance exercise (Blomstrand et al., 1995, 1997). It is suggested that BCAA supplementation may help to counteract the effects of an increase in plasma free tryptophan, and may thus improve the status of patients during or after some clinically stressful conditions.

Effect of vitamin B6 supplementation in McArdle's disease: a strategic case study.

A patient-blind study into the effect of a 10-week cessation of long-term vitamin B6 supplementation on B6 status and performance in McArdle's disease is reported. Muscle performance was assessed both subjectively and objectively by an ischaemic fatiguing protocol of the adductor pollicis muscle. Nine weeks after withdrawal of supplementation, vitamin B6 status had changed from adequacy to inadequacy and the force loss during the ischaemic fatiguing protocol had increased at all frequencies studied. The patient reported decreased exercise tolerance after 7 weeks and by the tenth week was experiencing an increase in muscle cramps. Vitamin B6 status and muscle performance may be linked in McArdle's disease and there is potential for enhancement of performance by B6 supplementation.

In vivo model of muscle pain: quantification of intramuscular chemical, electrical, and pressure changes associated with saline-induced muscle pain in humans.

Intramuscular injection of hypertonic saline is a good model to study human muscle pain (Kellgren 1938). The present study concerns the intramuscular (i.m.) pain mediators in saline-induced muscle pain. In experiment 1, the diffusion of infused hypertonic and isotonic saline (0.5 ml) in m. tibialis anterior was illustrated by magnetic resonance imaging (MRI) in one subject. In experiment 2, six volunteers received four sequential infusions (0.5 ml given at 5 min intervals) of isotonic saline and thereafter four sequential infusions (0.5 ml given at 5 min intervals) of hypertonic saline into m. tibialis anterior. The isotonic and hypertonic saline infusions were computer-controlled and separated by 20 min. The muscle pain intensity was assessed by continuous recordings on a visual analogue scale (VAS). One microdialysis probe was inserted 1 cm from the infusion needle in m. tibialis anterior and another probe in the other m. tibialis anterior. Concentrations of the i.m. sodium, potassium, magnesium, and prostaglandin E2 (PGE2) were assessed from the dialysates. Intramuscular electromyography (EMG) and pressure were assessed in the area of the infused saline. In experiment 1, the infusion of hypertonic and isotonic saline created a visible saline-pool on the MRI scans. These saline-pool volumes were stable and not correlated to the pain scores. In experiment 2, infusion of isotonic saline produced little pain compared to infusion of hypertonic saline. Maximal pain was reported after the first infusion of hypertonic saline and thereafter the pain gradually decreased with subsequent infusions of hypertonic saline. During infusion of hypertonic saline the i.m. sodium and potassium concentrations increased significantly, i.m. magnesium concentration tended to be increased, and the i.m. PGE2 concentration tended to be decreased although these changes were not significant. The i.m. EMG was smaller during and after infusions of hypertonic saline compared with isotonic saline. The i.m. pressure was not different during the infusions of hypertonic and isotonic saline but was increased between the infusions of hypertonic saline. This study has shown that i.m. infusion of hypertonic saline produced a saline-pool, causing the i.m. pressure to increase. Possibly, pain activation and cessation are related to increased intramuscular sodium and potassium content respectively.

Objective quantification of muscle and fat in human dystrophic muscle by magnetic resonance image analysis.

Information about changes in muscle composition has to date been primarily restricted to histological examination of biopsy samples or qualitative assessment of images obtained using a variety of techniques (e.g., ultrasound, CT, and MRI). We describe the development of a quantitative method for the analysis of muscle composition using MR T2 relaxation time mapping and image analysis. This approach provides an objective means of studying muscle and, when used in conjunction with force production measurements, may provide an accurate measure of response to muscle therapy.

McArdle's disease: molecular genetics and metabolic consequences of the phenotype.

McArdle's disease is defined as a lack of functional muscle glycogen phosphorylase. Analysis of the myophosphorylase gene has demonstrated substantial heterogeneity in the mutations that cause the disease, but in almost all individuals, the molecular phenotype is the absence of the protein in skeletal muscle. Muscle glycogen phosphorylase is a major repository of vitamin B6 in the body, accounting for at least 80% of the total body pool. In McArdle's patients, this pool is therefore missing, introducing the possibility that vitamin B6 metabolism might be altered in these individuals. Preliminary data have shown that McArdle's patients show signs of a subclinical vitamin B6 deficiency, and that oral vitamin B6 supplementation can improve vitamin B6 status and enhance fatigue resistance in muscle.

Immunohistochemical and biochemical indicators of muscle damage in vitro: the stability of control muscle and the effects of dinitrophenol and calcium ionophore.

The biochemical, histological and ultrastructural effects of 2,4-dinitrophenol and the calcium ionophore, A23187, on rat soleus muscle incubated in vitro have been examined to test the hypothesis that immunohistochemical techniques can be used to recognize early structural features of fibre damage. In control muscles, despite mild glycogen depletion and a mild reduction in protein synthetic rate in the central portion of the muscle, fibres throughout the muscle appear to be viable with normal cytoskeletal and contractile protein architecture, normal concentrations of high energy phosphates and no creatine kinase efflux. Dinitrophenol causes rapid creatine kinase efflux, extensive loss of immunolabelling for desmin and dystrophin, and abnormal myosin immunolabelling. Creatine kinase efflux and the changes in desmin and dystrophin are reduced by the exclusion of calcium. A23187 causes more gradual creatine kinase efflux associated with changes in myosin immunolabelling, but loss of desmin and dystrophin immunolabelling is restricted to a few of the most peripheral fibres. The results suggest that immunohistochemical methods can be used to reveal differences in the intracellular mechanisms of muscle damage. Although both dinitrophenol and A23187 may act, in part, through calcium-mediated processes, their effects on cytoskeletal proteins differ. Creatine kinase efflux after A23187 may not be due to gross sarcolemmal damage.

Developmental and gestational changes of phosphoethanolamine and taurine in rat brain, striated and smooth muscle.

Concentrations of taurine and phosphoethanolamine in rat smooth (intestinal and uterine), skeletal and cardiac muscle, and brain have been determined, using high-performance liquid chromatography (HPLC), to examine possible interrelationships in their tissue content. Concentrations were determined in fetal and neonatal samples, as well as in adult tissue, to investigate whether phosphoethanolamine and taurine levels are influenced by developmental state. The effect of gestational state was also studied. A marked decrease in cerebral phosphoethanolamine concentration during development was found together with a concomitant decrease in striated muscle but not in the two smooth muscles studied. A rise in uterine phosphoethanolamine during the early postpartum period confirmed previous NMR data. This occurred only in the uterus, suggesting it is specific to the process of involution within the myometrium. Taurine concentrations showed no consistent pattern of change with postnatal development. In adult animals, the highest levels of taurine were found in cardiac muscle. Pregnancy was associated with a fall in taurine concentration in all tissues, suggesting an influence of steroid hormones. As taurine is cotransported with Na+ in many systems it may be related to the increased water retention seen in pregnancy. It is concluded that marked changes in phosphoethanolamine and taurine levels occur during development and gestation, but that the changes are not interdependent, i.e., the changes are tissue specific.

Changes in human and rat uterine phosphoethanolamine and taurine with pregnancy and parturition.

Phosphoethanolamine and taurine have been identified in uterine extracts from rat and, for the first time, human tissue. The concentration of phosphoethanolamine [PEtn] determined by HPLC and 31P NMR spectroscopy, increased markedly (40%) within the first 6-12 h following parturition in rats, suggesting a role in involution. It appears that changes in [PEtn] account for the changes in the NMR phosphomonoester peak previously reported. A significant fall in [PEtn] was found in the human myometrium with pregnancy. Taurine was found in high concentrations in the rat and human uterus but decreased with pregnancy. The possible functional significance of these changes is discussed.

Effects of oxygen and antioxidants on the mitochondrial Ca-retention capacity.

2-Oxoglutarate-supported rat liver mitochondria were loaded with moderate amounts of calcium and submitted to O2 deprivation and reoxygenation. In the presence of acetoacetate, anaerobic energy production maintained Ca2+ retention by mitochondria during the anoxia period unless the Pi concentration of the incubation solution was raised to 4-6 mM. Acetoacetate prompted Ca2+ release from O2-deprived mitochondria at elevated Pi levels, presumably due to occurrence of a permeability transition of the inner membrane. Providing 3-hydroxybutyrate and malate, together with acetoacetate, was found to delay the permeability transition until O2 was reintroduced, i.e., O2 triggered a paradoxical release of Ca2+ from mitochondria under these conditions. Whether initiated by O2 in the presence of Pi or by Pi under aerobic conditions, Ca2+ release was initially activated and subsequently inhibited or reversed in the presence of alpha-tocopherol (10-90 mumol.g protein-1). Similar effects were exerted by alpha-tocopherol during Pi-induced Ca2+ release from oligomycin-treated mitochondria supported by succinate (+ rotenone). In addition, the permeability transition was delayed by retinol (3-30 mumol.g protein-1) while beta-carotene, ubiquinone, and water-soluble antioxidants, including Trolox C, were ineffective. Other observations suggest that the Ca(2+)-releasing and/or -retaining effects of alpha-tocopherol and retinol may be independent from pro- and/or antioxidant activities. Effects resembling those of alpha-tocopherol were exerted by alpha-tocopherol succinate, which is devoid of antioxidant activity. Our data indicate that the permeability transition of Ca(2+)-loaded liver mitochondria may be triggered by O2, in the presence of ketone bodies, and affected by lipid-soluble antioxidants through mechanisms seemingly unrelated to free-radical generation or scavenging.

Changes in frequency and force production of the human myometrium with alteration of pH and metabolism.

Changes in uterine metabolism and intracellular pH occur with uterine activity and these may in turn alter function. We have investigated the effects of changes in intracellular pH and metabolic inhibition on the spontaneous activity of isolated nonpregnant and pregnant human myometrial strips at 37 degrees C. Intracellular alkalinization resulted in a marked increase in the frequency of contractions in both nonpregnant and pregnant preparations. Intracellular acidification reduced force production in pregnant and nonpregnant preparations. The effects of pH were concentration dependent. Metabolic inhibition (2 mmol cyanide l-1) abolished spontaneous contractions in nonpregnant preparations (ten of ten) but pregnant preparations were able to maintain activity, although at a reduced level (eleven of fifteen). Possible mechanisms involved and relevance to uterine dysfunction are discussed.

The effect of vitamin E analogues and long hydrocarbon chain compounds on calcium-induced muscle damage. A novel role for alpha-tocopherol?

Previous studies have demonstrated that supplemental alpha-tocopherol inhibited calcium-induced cytosolic enzyme efflux from normal rat skeletal muscles incubated in vitro and suggested that the protective action was mediated by the phytyl chain of alpha-tocopherol [1]. In order to investigate this further a number of hydrocarbon chain analogues of tocopherol (7,8-dimethyl tocol, 5,7-dimethyl tocol, tocol, alpha-tocotrienol, alpha-tocopherol [10], vitamin K1, vitamin K1 [10], vitamin K1 diacetate, vitamin K2 [20], phytyl ubiquinone and retinol) were tested for any ability to inhibit calcium ionophore, A23187, induced creatine kinase (CK) enzyme efflux. Some compounds were found to be very effective inhibitors and comparison of their structures and ability to inhibit TBARS production in muscle homogenates revealed that the effects did not appear related to antioxidant capacity or chromanol methyl groups, but rather the length and structure of the hydrocarbon chain was the important mediator of the effects seen.

Effects of calcium ionophore on vitamin E-deficient rat muscle.

Damage to skeletal muscles may be mediated via free radicals or intracellular calcium overload. To look for inter-relationships between these pathways we have examined the effect of intracellular Ca overload on muscles from rats fed on either a vitamin E-deficient or vitamin E-sufficient diet and assessed the non-enzymic lipid peroxidation in these muscles by examining the production of thiobarbituric acid reactive substances by homogenates. Vitamin E-deficient muscles were more susceptible to Ca-induced intracellular enzyme efflux and this was acutely corrected by supplementation of the external medium with 230 mumol alpha-tocopherol/l. Vitamin E-deficient muscles showed increased levels of basal lipid peroxides and were more susceptible to iron-catalysed lipid peroxidation. Addition of the Ca ionophore A23187 increased lipid peroxidation in vitamin E-deficient muscle homogenates, but had the opposite effect in vitamin E-sufficient muscles. These results demonstrate that vitamin E-deficient muscle has an increased susceptibility to intracellular Ca overload, but that this effect cannot be explained by a direct stimulatory effect of the ionophore on non-enzymic lipid peroxidation.

Inhibition of Ca2+-induced cytosolic enzyme efflux from skeletal muscle by vitamin E and related compounds.

1. Efflux of an intracellular enzyme (creatine kinase) from normal rat skeletal muscles was induced by treatment with the Ca2+ ionophore A23187. Addition of alpha-tocopherol (230 microM) to the incubation medium was found to significantly diminish this efflux, and this effect was mimicked by alpha-tocopherol acetate, phytol and isophytol, but not by Trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). 2. Analysis of muscle cation content has shown that these protective effects of alpha-tocopherol etc. are not due to an inhibition of the Ca2+ accumulating effects of the ionophore. 3. Non-enzymic lipid peroxidation of skeletal-muscle homogenates was found to be inhibited by alpha-tocopherol and Trolox C, partially inhibited by phytol and isophytol, but unaffected by alpha-tocopherol acetate. 4. The activity of lipoxygenase enzymes was partially inhibited by alpha-tocopherol, phytol and isophytol, but not by alpha-tocopherol acetate or Trolox C. 5. Prostaglandin E2 efflux from isolated skeletal muscles was stimulated by treatment with the Ca2+ ionophore, but this was unaffected by alpha-tocopherol treatment.