Abnormal reinnervation of skeletal muscle in a tenascin-C-deficient mouse.

The possible involvement of tenascin-C in the reinnervation of a skeletal muscle was investigated in the tenascin-C-deficient mouse (T-/-) produced by Saga et al. (1992; Genes Dev 6:1821-1831). The pattern of reinnervation, observed after denervation of the triangularis sterni muscle, differs in T-/- and wild-type muscles in several traits. Axonal growth and stability of terminal arbors are impaired in the T-/- muscle: Some axons in mutant muscles grow beyond their original targets and reinnervate other synaptic sites, which may become dually innervated. In contrast to wild type, polyinnervation increases with time after denervation in T-/- muscles and is still present 7 months after nerve crush. The expression of a tenascin-C mRNA product disappears between 1 and 2 months after nerve crush. Of interest is that this transcriptional regulation in T-/- muscles occurs when major alterations in the morphology of regenerating endings become obvious. These observations strongly implicate tenascin-C in the formation, maturation, and stabilization of the neuromuscular junction.

Enhanced neurotransmitter release is associated with reduction of neuronal branching in a Drosophila mutant overexpressing frequenin.

Frequenin is a Drosophila Ca2+ binding protein whose overexpression causes a chronic facilitation of transmitter release at the larval neuromuscular junction and multiple firing of action potentials. These functional abnormalities are similar to those found in other hyperexcitable mutants (Shaker, ether-a-gogo, Hyperkinetic) which, in turn, exhibit increased branching at the motor nerve endings. We report here that mutants which overexpress frequenin have motor nerve terminals with reduced number and length of branches as well as number of synaptic boutons. Similar defects are observed in transgenic flies which have additional copies of the frequenin gene indicating that the phenotype can be adscribed to the overexpression of the protein. The ultrastructure of boutons, however, appears indistinguishable from wild type. In addition, we show here that frequenin overexpression leads also to a down regulation of Shaker proteins expression. The contrast between the observations in frequenin and the other hyperexcitable mutants indicates that nerve terminal morphology and enhanced transmitter release do not have a direct causal relationship.

Incorporation of synaptotagmin II to the axolemma of botulinum type-A poisoned mouse motor endings during enhanced quantal acetylcholine release.

The involvement of terminal sprouts in neurotransmitter release by in vivo botulinum type-A toxin poisoned motor endings was investigated 15 to 40 days after a single injection of the toxin onto the levator auris longus muscle of the mouse. Enhanced quantal acetylcholine release was induced by alpha-latrotoxin or La3+ in conditions that prevent endocytosis, and an antibody directed against the lumenal domain of synaptotagmin II (Syt II) was used in the presence or absence of Triton X-100. We showed that, under resting conditions, the intravesicular domain of Syt II requires Triton X-100 to be labelled, whereas it becomes exposed to the outside of the axolemma of both the original terminal arborization and the newly formed sprouts during enhanced exocytosis. These data were taken to indicate that, when sprouting is prominent, the whole modified terminal arborization, including the original branches and the sprouts, possesses the machinery for Ca2+-independent exocytosis.

The peripheral nerve and the neuromuscular junction are affected in the tenascin-C-deficient mouse.

A thorough examination of the structure and plasticity of the neuromuscular system was performed in tenascin-C mutant mice deficient in tenascin-C. The study of the peripheral nerve revealed a number of abnormal features. In the motor nerve, numerous unmyelinated and myelinated fibers with degraded myelin were present. Schwann cell processes often enclosed degenerative terminals. Transgene (beta-galactosidase) expression analyzed at the ultrastructural level was found to be unequally distributed in the mutant's neuromuscular tissues. At the NMJ, preterminal disorganization was prevalent. Some axon terminals exhibited abnormal overgrowth. A surprising lack of beta-galactosidase expression at some cellular sites known to possess tenascin-C in wild type mice correlated best with marked changes in the cytoarchitecture of the peripheral nerve and NMJ. In some other -but not all- cellular sites which normally express the molecule, immunofluorescence analysis suggested the presence of significant but low levels of tenascin-C-like immunoreactivity together with beta-galactosidase expression. Messenger RNA detection by RT-PCR confirmed the presence of low amounts of tenascin-C mRNA in skeletal muscle suggesting that the mice deficient in tenascin-C are not complete knock-outs of this gene, but low-expression mutants. Following in vivo injections of botulinum type-A toxin, we observed a greatly reduced sprouting response of the motor nerves in tenascin-C mutant mice. We also observed that N-CAM and beta-catenin were overexpressed in the mutant. Our results suggest that tenascin-C is involved both in stabilization and in plasticity of the NMJ.

Nitric oxide and peroxynitrite affect differently acetylcholine release, choline acetyltransferase activity, synthesis, and compartmentation of newly formed acetylcholine in Torpedo marmorata synaptosomes.

Recent reports proposed that nitric oxide was a modulator of cholinergic transmission. Here, we examined the role of NO on cholinergic metabolism in a model of the peripheral cholinergic nervous synapse: synaptosomes from Torpedo electric organ. The presence of NO synthase was immunodetected in the cell bodies, in the nerve ending area of nerve-electroplate tissue and in the electroplates. Exogenous source of NO was provided from SIN1, a donor of NO and O2-., and an end-derivative peroxynitrite (ONOO-). SIN1 increased calcium-dependent acetylcholine (ACh) release induced by KCl depolarization or a calcium ionophore A23187. The formation of ONOO- was continuously followed by a new chemiluminescent assay. The addition of superoxide dismutase, that decreases the formation of ONOO-, did not impair the stimulation of ACh release, suggesting that NO itself was the main stimulating agent. When the endogenous source of NO was blocked by proadifen, an inhibitor of cytochrome P450 activity of NO synthase, both KCl- and A23187-induced ACh release were abolished; nevertheless, the inhibitor Ng-monomethyl-L-arginine did not modify ACh release when applied in a short time duration of action. Both NO synthase inhibitors reduced the synthesis of ACh from the radioactive precursor acetate and its incorporation into synaptic vesicles as did ONOO- chemically synthesized or formed from SIN1. In addition, choline acetyltransferase activity was strongly inhibited by ONOO- and SIN1 but not by the NO donors SNAP and SNP or, by NO synthase inhibitors. Altogether these results indicate that NO and ONOO modulate presynaptic cholinergic metabolism in the micromolar range, NO (up to 100 microM) being a stimulating agent of ACh release and ONOO- being an inhibitor of ACh synthesis and choline acetyltransferase activity.

Nerve terminal sprouting in botulinum type-A treated mouse levator auris longus muscle.

The marked outgrowth of the motor nerve terminal arborization triggered by an in vivo local injection of Clostridium botulinum type-A toxin in the mouse levator auris longus muscle was studied with morphological and immunochemical approaches. The increase in total nerve terminal length depended on the time elapsed after toxin administration and was due to both increased number of terminal branches and branch length as revealed by a quantitative morphological analysis of whole mounts using the combined cholinesterase-silver stain. Nerve terminal sprouts increased in number, length and complexity even after the functional recovery of neuromuscular transmission had occurred as revealed by electrophysiological examination. Although we cannot exclude that transmitter release sites from the original nerve terminal arborization may still be functional after botulinum type-A toxin (BoTx-A) treatment, it is likely that newly formed functional release sites on the sprouts play a major role in the functional recovery of neuromuscular transmission. The presence of an immunoreactivity to synaptophysin and synaptotagmin-II, integral proteins of synaptic vesicles, gives support to our previous findings suggesting that nerve terminal sprouts have the molecular machinery for acetylcholine release.

Synaptotagmin II immunoreactivity in normal and botulinum type-A treated mouse motor nerve terminals.

The distribution of synaptotagmin II, a synaptic vesicle protein, was examined by immunohistochemistry at normal mouse motor nerve terminals and after botulinum type-A treatment. An immunoreactivity to synaptotagmin II was detected in both control and botulinum type-A treated motor nerve terminals including newly formed sprouts. These data, together with other reports showing the absence of synaptotagmin I at the neuromuscular junction, suggest that synaptotagmin II is the isoform involved in transmitter release at motor nerve terminals.

Upregulation of calcitonin gene-related peptide at mouse motor nerve terminals poisoned with botulinum type-A toxin.

Calcitonin gene-related peptide (CGRP)-like immunoreactivity of motor nerve terminals was investigated at different times after local in vivo injection of botulinum type-A toxin (BoNT/A) close to the mouse levator auris longus muscle. CGRP expression in most of control nerve terminals was undetectable, but markedly increased during muscle paralysis and synaptic remodelling and, declined once functional recovery occurred.

Mouse motor nerve terminal immunoreactivity to synaptotagmin II during sustained quantal transmitter release.

An antibody directed against the lumenal NH2-terminus of synaptotagmin II was used to examine the distribution of this vesicular protein either after spontaneous acetylcholine release or after sustained release induced by La3+ or alpha-latrotoxin, in conditions that prevent endocytosis. The detection of the epitope was examined in the presence or absence of Triton X-100. We show that, in resting conditions of transmitter release, permeabilization of nerve terminal membranes is required for obvious detection of synaptotagmin Ii immunoreactivity whereas during sustained rates of quantal release, permeabilization is not necessary. These data indicate that, in the latter conditions, synaptotagmin II is incorporated into the terminal axolemma and its intravesicular domain exposed at the extracellular nerve terminal surface.

Plasticity of motor nerve terminals in Drosophila T (X,Y)V7 mutant: effect of deregulation of the novel calcium-binding protein frequenin.

The Drosophila T(X,Y)V7 mutant is characterized by abnormally large motor responses that build up upon repetitive stimulation. Genetically it is characterized by a chromosomal breakpoint located at the proximal end of the Shaker gene complex. This mutation affects a gene which encodes a novel calcium-binding protein: the frequenin. Since neuronal activity is known to affect neurite elongation we looked for the geometry of motor terminal arborization in this mutant. Our results show a significant reduction in number and length of motor terminal branches in mutants as compared to wild type. This observation is opposite to the effect of other hyperexcitable mutations such as Shaker or ether-a-gogo or Hyperkinetic. Thus the V7 phenotype cannot be interpreted as a result of changes in motoneuron firing pattern. According to results obtained on transformed larvae in which frequenin cDNA expression was under the control of a heat shock promoter, it appears that the morphological phenotype of V7 may be due to specific effects of deregulation of this calcium-binding protein.

Sprouting of mammalian motor nerve terminals induced by in vivo injection of botulinum type-D toxin and the functional recovery of paralysed neuromuscular junctions.

Paralysis of the mouse levator auris longus muscle by in vivo injection of Clostridium botulinum type-D neurotoxin (BoNT/D) triggered a marked outgrowth of the motor nerve from the original terminal arborization. The increase in total nerve terminal length was due to both increase in the number of terminal branches and in average branch length. Asynchronous quantal transmitter release in response to nerve impulses was a prominent feature in paralysed junctions that started 24 h after poisoning and lasted for about 15 days. The functional recovery of poisoned junctions occurred 25-30 days after poisoning and was characterized by the synchronous quantal transmitter release upon nerve stimulation that triggered synaptically evoked action potentials and muscle fibre contraction.

Divalent cation changes in cerebellar Purkinje cells after climbing fiber deafferentation.

A secondary ion mass spectrometry (SIMS) microscope was used to detect intracellular stores of calcium, magnesium, sodium and potassium. Measurements were made in semithin sections of fixed tissues of normal and climbing fiber deafferented cerebellar cortex. Quantitative data were collected from 150 microns diameter image fields in the molecular and granule layers. The results indicate smaller quantities of both calcium and magnesium in the deafferented cerebellar cortex compared to the normals, the molecular as well as the granule layer being affected. The results are discussed in terms of the usefulness and limitations of the SIMS microscope for histological preparations.

Validity of large scale standardised behavioural screening.

A nation-wide screening programme in the Netherlands is described. 86% of all children are invited at the age of 9 months for the three-stage Ewing screening: 94% participate. The screening procedure is standardized in very great detail, including test, registration, training and guidance. The objective of the screening programme is to detect moderate to severe sensorineural hearing loss as well as long-standing conductive hearing loss. Tympanometry studies clearly show that the selection process of conductive hearing loss is associated with otitis media with effusion (OME) lasting 3 months or more. Studies on two cohorts of children with sensorineural hearing loss show test standardization and training to be vital elements of behavioural screening. All studies show validity parameters of 85% or more.

Presynaptic actions of botulinal neurotoxins at vertebrate neuromuscular junctions.

1. In the present paper we review some presynaptic aspects of the mode of action of botulinal toxins (BoTxs) at vertebrate neuromuscular junctions with emphasis on studies carried out in our laboratories using electrophysiological and morphological techniques. 2. Spontaneous quantal transmitter release recorded as miniature end-plate potentials is drastically affected by BoTxs. The low probability of release at poisoned terminals can be enhanced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), Cd2+ and La3+. However, CCCP and La3+ which drastically deplete clear synaptic vesicles from unpoisoned terminals failed to markedly affect the density of synaptic vesicles at poisoned terminals. It is concluded that poisoned terminals have a reduced sensitivity to the release-promoting action of Ca2+, Cd2+ and La3+. 3. When comparing the effect of the various BoTxs on nerve-impulse evoked transmitter release it appears that increasing phasic Ca2+ entry into the terminals enhances evoked synchronized quantal release only from terminals poisoned with serotypes A and E. In contrast, enhanced Ca2+ entry into terminals poisoned with serotypes B, D and F induced a period of high frequency asynchronous release suggesting that these BoTxs may affect a presynaptic step beyond the influx of Ca2+, that may be involved in the synchronization of transmitter quanta. These data suggest that the actions of BoTxs involve several steps of the acetylcholine release process. 4. The analysis of presynaptic currents which depend on both Ca2+ entry and intraterminal background Ca2+ levels strongly suggests that neither Ca2+ entry nor intraterminal Ca2+ levels are altered by BoTxs. Furthermore, poisoned terminals are no more efficient than unpoisoned ones in dealing with Ca2+ overloads. 5. Finally, the morphological examination of junctions paralysed by BoTx-A indicates that the toxin triggers a particularly important overgrowth of the nerve terminals and suggests that the in vivo functional recovery may occur from an extension of the original nerve terminal arborization and the concomitant remodelling of postsynaptic structures.

Terminal sprouting in mouse neuromuscular junctions poisoned with botulinum type A toxin: morphological and electrophysiological features.

Functional properties of terminal sprouts elicited by an in vivo injection of Clostridium botulinum type A toxin were studied in endplates of the Levator auris longus muscle of the mouse poisoned from a few days to 28 days beforehand. For this purpose, morphological observations of the extent of terminal sprouts and localization of acetylcholine receptors was performed in whole mount preparations. Sprouts appeared as thin unmyelinated filaments that run usually parallel to the longitudinal axis of the muscle fibres; labelling acetylcholine receptors revealed their line-shaped accumulation co-localized with the sprouts. In addition, presynaptic membrane currents elicited by nerve stimulation were recorded by external electrodes applied under visual control onto the membrane of pre-existing motor endings and newly formed sprouts. These recordings showed the presence of widespread triphasic waveforms which indicated active impulse propagation of the action potential over most of the length of the poisoned endings. Ca2+ influx and Ca2(+)-dependent K+ currents in the sprout membrane were found to be similar to those described in unpoisoned endings. The presence of normal Ca2+ influx, upon active depolarization, in the terminal sprout membranes together with the localization of acetylcholine receptors in front of these membranes, indicates that the terminal sprouts may play a role in the recovery of neuromuscular transmission after Clostridium botulinum poisoning.

Presynaptic calcium currents recorded from calyciform nerve terminals in the lizard ciliary ganglion.

Electron microscopic examination of ciliary ganglion from Anolis carolinensis shows large calyciform presynaptic nerve terminals ending on ganglion cells. Intracellular records were obtained from the terminals, and membrane currents were recorded with the single-electrode voltage clamp technique. After block of Na+ currents with tetrodotoxin, depolarization of the terminals produced inward currents that disappeared when extracellular Ca2+ was removed, and increased in magnitude and duration when competing outward currents were blocked by intracellular Cs+. Thus it is possible, with this preparation, to record and characterize Ca2+ currents presumably associated with neurotransmitter release.

The levator auris longus muscle of the mouse: a convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins.

We describe here the levator auris longus muscle of the mouse as a convenient neuromuscular preparation for the in vitro study of presynaptic effects of drugs and toxins applied in vivo in young or adult mice. The good visibility of its motor axons and terminals using Nomarski optics allows accurate electrophysiological studies of presynaptic signals. In addition, the levator auris longus muscle is sufficiently thin to be stained as a whole mount preparation. Preliminary results indicate that some correlation can be established between changes in time course of the presynaptic signal and the morphology of motor endings after poisoning the levator auris longus muscle with botulinum type A toxin.

Inability of regenerating mouse motor axons to innervate a denervated target.

We used the triangularis sterni muscle of the adult mouse to study the influence of the connective sheaths that surround a nerve on the regeneration of its crushed axons. Our data indicate that regenerating axons: invade all the branches of a denervated pathway, independent of the presence of endplates; but are unable to escape from the perineurium even when vacant endplate sites are present in the immediate vicinity. Although regenerating axons do not share the first feature with intact sprouting axons, the second feature is common to both.

[Screening for hearing disorders. Current status and bottlenecks]. / Opsporing van gehoorstoornissen. Huidige stand van zaken en knelpunten.

In The Netherlands a nation-wide screening program on hearing for infants is available. This program started around the late sixties and is now incorporated in the Health Care System and added to the tasks of the Well Baby Clinics. A modification of the method as developed by the Ewings is used. This method is relatively easy to learn. It is, however, essential that it is executed accurately and with a maximum of care. Therefore the testing itself as well as the assessment of the results is strictly standardized and a lot of attention is given to the training program. The Dutch Foundation for the Deaf and Hearing-Impaired Child co-ordinates the screening program. About 75% of the 9 months old babies are screened and some 5% of the children has to be referred. The Ewing-test has proved to be effective. It detects both severe and moderate sensory-neural hearing-losses as well as conductive hearing-losses. However, the program faces us with a few difficulties: the structure is effective, though time-consuming; the referral-procedure is not always satisfactory. Due to different factors, some groups of children, sometimes at risk, are not covered by the screening program or any other program.