Inhibin B and anti-Müllerian hormone/Müllerian-inhibiting substance may contribute to the male bias in autism.

The autistic spectrum disorders have a significant male bias in incidence, which is unexplained. The Sertoli cells of the immature testes secrete supra-adult levels of Müllerian-inhibiting substance/anti-Müllerian hormone (AMH) and inhibin B (InhB), with both hormones being putative regulators of brain development. We report here, that 82 boys with an autism spectrum disorder have normal levels of InhB and AMH. However, the boys' level of InhB correlated with their autism diagnostic interview-revised (ADI-R) scores for the social interaction (R=0.29, P=0.009, N=82) and communication domains (R=0.29, P=0.022, N=63), and with the number of autistic traits the boys exhibited (R=0.34 and 0.27, respectively). The strengths of the abovementioned correlates were stronger in the boys with milder autism (R=0.42 and 0.50, respectively), with AMH exhibiting a significant negative correlation to the ADI-R score in these boys (R=-0.44 and R=-0.39, respectively). Neither hormone correlated to the incidence of stereotyped and repetitive behaviours. This suggests that the male bias in the autistic spectrum has multiple determinants, which modulate the effects of an otherwise non-dimorphic pathology. Furthermore, AMH and InhB have opposing effects on the SMAD1/5/8 pathway, and opposing correlates to autistic traits, implicating the SMAD pathways as a putative point of molecular convergence for the autistic spectrum.

The male bias in the number of Purkinje cells and the size of the murine cerebellum may require Müllerian inhibiting substance/anti-Müllerian hormone.

There is a male bias in the size of the cerebellum, with males, on average, having more Purkinje cells than females. The critical periods in cerebellum development occur when the immature testes secrete Müllerian inhibiting substance (MIS; synonym anti-Müllerian hormone) but only trace levels of testosterone. This suggests that the male bias in the cerebellum is generated by a different mechanism to the testosterone-sensitive reproductive nuclei. Consistent with this, in the present study, we report that Purkinje cells and other cerebella neurones express receptors for MIS, and that MIS(-/-) male mice have female-like numbers of Purkinje cells and a female-like size to other parts of their cerebellum. The size of the cell bodies of Purkinje cells was also dimorphic, although only a minority of this was a result of MIS. This suggests that MIS induces the initial male bias in the cerebellum, which is then refined by pubescent testosterone and/or other sex-specific factors.

Analysis of mRNAs that are enriched in the post-synaptic domain of the neuromuscular junction.

The identity of synaptically-enriched genes was investigated by comparing the abundance of various mRNAs in the synaptic and extra-synaptic regions of the same muscle fibers. The mRNAs for several known synaptic proteins were significantly elevated in the synaptic region when measured by real-time PCR. The synaptic mRNAs were then further analyzed using microarrays and real-time PCR to identify putative regulators of the neuromuscular junction (NMJ). MRF4 was the only member of the MyoD family that was concentrated at the mature NMJ, suggesting that it may have a unique role in the maintenance of post-synaptic specialization. Three potential regulators of the NMJ were identified and confirmed by real-time PCR: glia maturation factor gamma was concentrated at the NMJ whereas Unr protein and protein tyrosine phosphatase were repressed synaptically. The identification of synaptically-repressed genes may indicate that synaptic specialization is created by a combination of positive and negative signals.

The use of sodium lamps to brightly illuminate mouse houses during their dark phases.

The human and murine diurnal rhythms are out of phase. Consequently in conventionally-lit mouse houses the mice's deep sleep is often disrupted, the daily welfare monitoring of the mice is limited by their inactivity, and scientific data obtained from the mice model the sleeping rather than awake human. Sodium light is bichromatic, with both wavelengths being in the human visual field but at the margin of murine vision. We report here that sodium lamps can be used to light mouse houses to a level that is comfortable for humans, but still sufficiently dull to permit nocturnal behaviour in mice. The response of mice to sodium light was initially monitored by recording the locomotory activity of BALB/c mice. The movement of mice in their cages greatly increased at the start of the nocturnal phase. Alterations in the white light cycle caused an acute change in the onset of nocturnal behaviour. In contrast, sodium light did not suppress the onset of nocturnal locomotory behaviour, even though the lighting was sufficiently bright for humans to read without light adaptation. The sodium lighting was then used to observe the nocturnal behaviour of over 150 mice of various strains, for over 1.5 years. Mice were invariably awake and alert during the nocturnal/sodium light phase. All exhibited high locomotory activity, except for nursing mothers. Some tasks, such as cage cleaning and minor surgery, were more easily done under white than sodium lighting. We therefore adjusted the timing of the light cycles to provide white light in the morning and sodium light (nocturnal phase) in the afternoon. This provided for easy operation of the mouse house, while yielding both animal welfare and scientific advantages.

Fibroblast growth factor-5 is expressed in Schwann cells and is not essential for motoneurone survival.

Fibroblast growth factor-5 (FGF-5) is a putative target-derived survival factor for motoneurones as it is concentrated in the synaptic portions of skeletal muscles and because it promotes the survival of embryonic motoneurones in vitro. A variety of experimental approaches have been used to examine this possibility. The expression of FGF-5 in the neuromuscular system was analysed using the reverse transcription-polymerase chain reaction (RT-PCR). Both splice variants of FGF-5 were detected in adult rat skeletal muscle, sciatic nerve, and spinal cord. The expression of FGF-5 in skeletal muscle was up-regulated after denervation. At first sight this appears to be consistent with FGF-5 being a target-derived factor. However, FGF-5 protein was detected in Schwann cells, macrophages, vascular smooth muscle and endothelial cells, but not in muscle fibres. The absence of FGF-5 in muscle fibres was confirmed by RT-PCR examination of isolated muscle fibres. Furthermore, FGF-5 protein was also not detected in denervated fibres, as would be expected for a neuronal survival factor. Denervation did however lead to up-regulation of FGF-5 in the Schwann cells of the distal nerve trunk. This may indicate that FGF-5 is either an autocrine regulator of Schwann cells or a Schwann cell-derived neurotrophic factor. The latter appears not to be the case for two reasons. First, the double-ligation technique was used to show that endogenous FGF-5 is not transported in motor axons. Second, stereological estimates of the number of motoneurones in an FGF-5 null mutant (Angora) mouse failed to reveal any loss of motoneurones. Collectively these experiments suggest that FGF-5 is not a physiological regulator of motoneurones, and therefore raise the possibility that it is an autocrine regulator of Schwann cells.

TGF-beta 2 attenuates the injury-induced death of mature motoneurons.

The distributions of transforming growth factor-betas (TGF-betas) and their receptors suggest that the TGF-betas regulate motoneuron survival. This hypothesis was tested by avulsing the hypoglossal nerve of adult rats and perfusing either TGF-beta 2 or vehicle adjacent to the hypoglossal nucleus. By 4 weeks, half of the avulsed motoneurons had died. Infusion of 6 ng of TGF-beta 2 adjacent to the avulsed motor nucleus caused a significant attenuation of this death. This dose of TGF-beta 2 is low compared to that used with GDNF or BDNF in previous studies of avulsed motoneurons, indicating that TGF-beta 2 may be one of the more potent survival factors for adult motoneurons. TGF-beta 2 was, however, unable to prevent or reduce the axotomy-induced down regulation of choline acetyltransferase. Other motoneuron survival factors also have a narrow-spectrum of actions, suggesting that the homeostasis of motoneurons is regulated by a cocktail of growth factors with distinct but partially overlapping actions.

The expression and structure of TGF-beta2 transcripts in rat muscles.

The transforming growth factor-beta2 (TGF-beta2) transcripts expressed in various tissues of rat were characterised by RT-PCR and the nucleotide sequence of the cDNAs determined. A transcript with an 84-nucleotide insert in the latency-associated peptide region, the long form, was found. The long form of TGF-beta2 was detected in the aorta, primary bronchus, uterus, heart, skeletal muscle, sciatic nerve and spinal cord but not in the intestine. The 3' untranslated region of TGF-beta2 contained several putative AU-rich elements and multiple polyadenylation sites, indicating post-transcriptional regulation of TGF-beta2 synthesis. The levels of TGF-beta2 transcripts were estimated using semi-quantitative RT-PCR. They were down-regulated during muscle development and up-regulated after denervation. The long form constituted approximately 6% of the total TGF-beta2 messages in skeletal muscle.

Anterograde axonal transport of glial cell line-derived neurotrophic factor and its receptors in rat hypoglossal nerve.

Glial cell line-derived neurotrophic factor is one of the most potent motoneuron survival factors yet identified. Although retrograde transport of trophic factors to the cell body is thought to be an important process in motoneuron survival, the transport pathways that lead to interaction of glial cell line-derived neurotrophic factor with its receptors is not known. We have used a double ligated hypoglossal nerve preparation to investigate transport of endogenous glial cell line-derived neurotrophic factor and its receptors, glial cell line-derived neurotrophic factor family receptor alpha1 and receptor re-arranged during transfection. Glial cell line-derived neurotrophic factor was found to accumulate at the distal ligature, indicating retrograde transport and consistent with its motoneuron survival effects. In addition, we observed accumulation of glial cell line-derived neurotrophic factor and its receptors at the proximal ligature, indicating anterograde transport. This finding is not predicted by neurotrophic theory. Staining for glial cell line-derived neurotrophic factor in the motor axons was punctate, suggesting involvement of transport vesicles. Results obtained using immunohistochemistry and reverse transcription-polymerase chain reaction provide evidence for the synthesis of glial cell line-derived neurotrophic factor and glial cell line-derived neurotrophic factor family receptor alpha1 in Schwann cells and glial cell line-derived neurotrophic factor family receptor alpha1 and receptor re-arranged during transfection in motoneuron cell bodies. When the motor axons were separated from the cell body by avulsion, glial cell line-derived neurotrophic factor remained in the vicinity of the Schwann cells and did not accumulate at the proximal ligature. Our results indicate anterograde transport of Schwann cell-derived glial cell line-derived neurotrophic factor, which is dependent on binding to its cell body-derived receptors. These findings suggest a mechanism for collection of glial cell line-derived neurotrophic factor from multiple Schwann cells which surround motor axons. We propose that in addition to its role in motoneuron survival, glial cell line-derived neurotrophic factor may also modulate local neuronal effects in distal regions of the nerve.

Transforming growth factor-beta 2 is anterogradely and retrogradely transported in motoneurons and up-regulated after nerve injury.

The survival of motoneurons is dependent on them receiving continual trophic support from muscle fibres and various other cell types. Numerous putative survival factors have been identified and a set of criteria established by which these candidates can be assessed. These criteria include the need for the factor and its receptors to be in appropriate locations and for the factor or its second message to be retrogradely transported. In this paper, we demonstrate that a multifunctional cytokine, transforming growth factor-beta 2, appears to meet these criteria. The locations of the transforming growth factor-beta 2 and its receptors in the neuromuscular system were determined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Motoneurons were shown to synthesize the three proteins involved in transforming growth factor-beta 2 signalling (types I and II transforming growth factor-beta receptor and betaglycan) and to transport them anterogradely, where they were inserted into the axonal membrane and nerve terminal. Transforming growth factor-beta 2 was detected in the synaptic portions of muscle fibres, motoneurons and in injured nerves, indicating that motoneurons may be exposed to multiple and potentially redundant sources of transforming growth factor-beta 2. Double-ligation experiments were used to demonstrate that motoneurons transport transforming growth factor-beta 2 up and down their axons. The anterograde transport of both transforming growth factor-beta 2 and its receptors, coupled with the fact that most of a motoneuron's mitochondria are located in the axon, raises the issue of whether the repression of the initiation of apoptosis is restricted to the cell body or occurs along the entire length of a neuron.

Development of skeletal muscles in transforming growth factor-beta 1 (TGF-beta1) null-mutant mice.

Fetal transforming growth factor-beta 1 (TGF-beta1) has been postulated to regulate the onset of myotube formation and/or pattern formation in developing skeletal muscles. In apparent contradiction of these hypotheses, the development of the extensor digitorum longus and soleus in TGF-beta1 null-mutant muscle was normal. The onset of secondary myotube formation, the numbers of myotubes formed, the proportion of fast and slow fibers, and the patterns of fiber types and connective tissues were essentially identical in TGF-beta1(+/+) and TGF-beta1(-/-) mice. A portion of the TGFbeta1 in skeletal muscles is derived from the mother, via the placenta. This maternal-derived TGF-beta1 was also not essential for the development of skeletal muscles, as the characteristics of pups born to a TGF-beta1(-/-) mother were normal TGF-beta1(-/-) mice die at weaning due to a generalized autoimmune attack. This postnatal death was circumvented by breeding the TGF-beta1 null mutation into nude mice (Whn(-/-)). Like many other strains of TGF-beta1(-/-) mice, extensive loss of Whn(-/-), TGF-beta1(-/-) embryos occurred in utero. However, a portion of the Whn(-/-), TGF-beta1(-/-) mice survived past weaning, remained healthy, and were fertile. The TGF-beta1(-/-) x Whn(-/-) mouse thus represents a valuable tool for the study of the function of TGF-beta1 in the adult, including its putative role as a pregnancy-related hormone.

The myotubal origin of rat muscle fibres affects the extent of tenotomy-induced atrophy.

1. Immature muscle fibres (myotubes) can be divided into primary and secondary generations, which differ from each other in their time of formation, growth rates and myosin isoform expression. It is unclear whether the intrinsic differences between primary and secondary myotubes are totally extinguished once they mature and extrinsic factors, such as load, become important. 2. Four pregnant rats were injected with 5-bromo-2'-deoxyuridine (BrdU) on the 14th and 15th days of gestation. This selectively and permanently labels primary myotubes. Ten rats from four litters were killed when 8 months old, with three males (365-430 g) from a single litter being used for the quantitative study and the remainder being examined qualitatively. The extensor digitorum longus muscle (EDL) in each rat was tenotomized for 14 days. The sizes of fibres in the EDL were then correlated with their fibre type and whether they contained BrdU-labelled nuclei. 3. We reported that (i) II A and II B fibres derived from primary myotubes atrophied significantly less after tenotomy than II A and II B fibres derived from secondary myotubes and (ii) BrdU-labelled myonuclei were retained in the tenotomized muscle, even though tenotomy resulted in a substantial loss of myonuclei from the EDL. 4. We conclude that the origin of a fibre is a determinant of its response to the external forces which control its size, and hence force generation.

Transforming growth factor-beta2 is elevated in skeletal muscle disorders.

The transforming growth factor betas (TGF-betas) are multifunctional growth factors that act on both fibroblasts and myosatellite cells. In rodent models of muscle diseases, high levels of TGF-beta2 are expressed by myogenic cells. We have examined whether the expression of TGF-beta2 is also elevated in diseased human muscles. The disorders examined were Duchenne muscular dystrophy, myotonic dystrophy, myotubular myopathy, spinal muscular atrophy, and amyotrophic lateral sclerosis. The levels of TGF-beta2 immunoreactivity were elevated in atrophic, necrotic, and regenerating fibers and in fibers with central nuclei or cytoplasmic masses, irrespective of whether fibrosis was present. We therefore suggest that TGF-beta2 is important for muscle repair and that the presence of a TGF-beta within a muscle only leads to fibrosis if certain other factors are present.

The non-synaptic expression of transforming growth factor-beta 2 is neurally regulated and varies between skeletal muscle fibre types.

In adult skeletal muscles, transforming growth factor-beta 2 is restricted to the postsynaptic domain of the neuromuscular junction. The various putative functions of this transforming growth factor-beta 2 predict different patterns of transforming growth factor-beta 2 expression in denervated muscles. We therefore denervated rat tibialis anterior, extensor digitorum longus and soleus muscles and examined the expression of transforming growth factor-beta 2 using semi-quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. Denervation up-regulated transforming growth factor-beta 2 expression extrasynaptically with little or no effect on synaptic expression. The up-regulation was detectable by one day, had become significant by three days and remained elevated for at least two weeks. This proves that the transforming growth factor-beta 2 associated with the neuromuscular junction is not under neural control and is consistent with transforming growth factor-beta 2 being a trophic factor for motoneurons. This pattern of transforming growth factor-beta 2 expression is similar to that described for other proteins associated with the neuromuscular junction, notably the acetylcholine receptor subunit genes. However, in contrast to the acetylcholine receptor subunit genes, the extent of up-regulation of transforming growth factor-beta 2 varied between fibre types, with the glycolytic IIB fibres being less affected than other fibre types.

Primary myotubes preferentially mature into either the fastest or slowest muscle fibers.

Myoblasts and myotubes are heterogeneous, but what is the significance of this heterogeneity? Is it a vital component of the mechanism by which a muscle develops or is it part of the process that generates mature fibers with diverse sizes, speeds of contracture, and metabolisms? We have begun to explore these questions by using BrdU to selectively label rat primary myotubes, thus enabling their mature characteristics to be defined for the first time. In the soleus, the type I fibers of primary myotube origin were 21% larger than those of secondary myotube origin, indicating that the origin of a fiber can affect its mature force production. In the extensor digitorum longus (EDL), the primary myotubes differentiated into all known fibers types, but with marked variation in frequency. In the superficial portion of the EDL, 97% of primary myotubes became IIB fibers, even though approximately 41% of the fibers in this region are IIA or IIX. In the deep portion, primary myotubes preferentially developed into type I fibers. Thus, primary myotubes in the EDL predominantly differentiate into the two most dissimilar fiber types: the slowest, smallest, most oxidative, type I fibers and the largest, fastest, most glycolytic, type IIB fibers. Each of the subtypes of primary myotubes had a different fate. In the EDL, the slow and fast primary myotubes appeared to differentiate into type I and IIB fibers, respectively. This implies that spatial and temporal signals operating in the limb are major determinants of the mature pattern of fiber types and that innervation of a muscle involves a selective matching between the various types of motoneurons and muscle fibers.

Accumulation of tau in autophagic vacuoles in chloroquine myopathy.

Long-term administration of chloroquine to rats induces a vacuolar myopathy, which is specifically termed chloroquine myopathy (CM). In CM, tau mRNA levels were transiently upregulated in the early phase, while tau itself slowly accumulated in the late phase. The temporal profiles of tau mRNA levels and its accumulation were very similar to those of beta-amyloid protein precursor (APP) and its carboxyl-terminal fragments, both of which have been known to be degraded in lysosomes. Immunocytochemistry showed that tau progressively accumulated in the rimmed vacuoles exhibiting increased acid phosphatase activities, and immunoelectron microscopy demonstrated that tau was located within autophagic vacuoles. These results suggest that the accumulation of tau in CM is due to defective tau degradation in the lysosomal compartment in the muscle.

The trophic requirements of mature motoneurons.

Mature motoneurons interact with many cell types, including skeletal muscle fibres, Schwann cells, glia and various neurons. Each of these cell types is thought to provide trophic support to motoneurons, but it is not known whether the support provided by one cell type can fully substitute for the absence of a signal from another cell type. The ability of various growth factors to support motoneurons in the absence of muscle fibres, Schwann cells or long-axon synaptic input has been studied using in vivo models. However, these studies do not define the total needs of motoneurons, as local spinal influences have not been removed. In this paper, the total trophic requirement of mature motoneurons was assessed by culturing them at a low cell density, in the absence of all other cell types. Under these conditions, mature motoneurons die by apoptosis within 24 h, which is equivalent to the rate at which immature motoneurons die in vitro. This is consistent with the emerging view that mature cells are primed for apoptosis. Nine putative trophic factors (BDNF, CNTF, FGF2, GDNF, IGF I, IGF II, NT3, NT4, TGF-beta2), either alone or in combination, were unable to prevent the rapid death of the cultured motoneurons, even though some of these factors are able to attenuate the affects of less severe injuries such as axotomy or avulsion. The survival of mature motoneurons may therefore be dependent on a combination of growth factors, with at least one of the factors being distinct from the above mentioned factors.

A murine specific splicing variant of the acetylcholine receptor epsilon-subunit gene transcript.

The acetylcholine receptor (AChR) is an ion channel involved in signal transmission from motorneuron to skeletal muscle. We describe here a murine-specific splicing variant of the epsilon subunit of the AChR (epsilon s), which lacks exon 5 epsilon s has a truncated extracellular domain which may affect the physiological characteristics of the resulting AChR channel.

Skeletal muscle fibre types: detection methods and embryonic determinants.

Muscle fibres used to be simply classified as either type I, IIa or IIb. Advances in molecular and histological techniques have, however, lead to the realisation that the phenotypes of muscles are more varied than this. An additional fibre type (IIX/IID) has been discovered, fibres with intermediate fibre types have been described and there is accumulating evidence that the fibres types described from the study of limb muscles are not necessarily applicable to other skeletal muscles, such as the jaw and extra-ocular muscles. Further to this has been the discovery that diversity occurs at all stages of muscle development. There are subpopulations of myoblasts and myotubes as well as various types of muscle fibres. The relationships between the different stages of development is still under study. However, it is clear that each stage of muscle development is influenced to a certain degree by prior events. Consequently, the characteristics of mature fibres reflect both their developmental origins and influences from the adult environment, such as their patterns of muscle activation.

Cellular localisation of transforming growth factor-beta 2 and -beta 3 (TGF-beta2, TGF-beta3) in damaged and regenerating skeletal muscles.

Regeneration involves a number of cellular processes: revascularisation, invasion by haemopoietic cells, removal of necrotic tissue and finally reformation of the tissues. These processes have been extensively studied in vitro and are known to be affected by various growth factors. However, it has proven difficult to extrapolate the in vitro results to the in vivo situation. This is partially because the response of cells to growth factors is dependent on which other regulatory factors are present. The locations of various growth factors within regenerating skeletal muscles have been studied but information is not available for the transforming growth factor-beta2 (TGF-beta2) or TGF-beta3, even though the TGF-betas are putative regulators of revascularisation, inflammation and the formation of connective tissue and muscle fibres. In this paper, the cellular locations of TGF-beta2 and TGF-beta3 in freeze-lesioned skeletal muscle were examined using immunohistochemistry. The amounts and locations of the TGF-betas varied depending on the stage of degeneration/regeneration. The first isoform of TGF-beta to appear within the lesion was TGF-beta2, which accumulated at the junctions between the viable and necrotic portions of fibres. The production of TGF-beta2 by the damaged fibres occurred immediately prior to the inflammatory reaction. However, these two events are probably independent of each other as the TGF-beta2-rich necrotic tissue was not preferentially phagocytosed. The haemopoietic cells contained TGF-beta3 immunoreactivity and the lesioned area became progressively rich in TGF-beta3 as the macrophages accumulated in the lesion and removed the TGF-beta2-rich necrotic tissue. In vitro, the TGF-betas are potent inhibitors of myogenic fusion and have been postulated to control the onset of myotube formation in vivo. Consistent with this idea, the formation of myotubes did not occur until the TGF-beta3-positive haemopoietic cells had migrated from the ghosts of necrotic fibres. In contrast, fusing satellite cells and newly formed myotubes contained strong TGF-beta2 immunoreactivity. This observation, coupled with the recent report that satellite cells require functional TGF-beta receptors to fuse in vivo, suggests that TGF-beta2 may stimulate myotube formation in vivo.