Size-related differences in the branching pattern of the motor nerve terminals in triangularis sterni muscle of the mouse

A light microscopy morphometric study was performed in singly innervated synaptic areas of the triangularis sterni muscle of the normal adult Swiss mouse. Investigating mechanisms of the motor nerve growth control, we tested the hypothesis that significant differences in the nerve terminal branching pattern can be detected between different populations of nerve endings classified according to their arborization complexity or size. The main observations of this morphometric study are first, that the mean segment length of the terminal arborization between branch points behaves as an independent variable from the remaining parameters; the mean value of this parameter did not change in nerve endings of differing size and complexity. Secondly, the increase in size of the nerve endings is accompanied by a significant reduction in the mean length of the distal free-end segments. Results are discussed in the context of the possible regulatory mechanisms governing nerve terminal growth and remodelling.     

Pattern of arborization of the motor nerve terminals in the fast and slow mammalian muscles.

A silver impregnation method and a morphometric approach were used to define differences existing in the motor nerve terminal branching pattern between a fast-twitch muscle (extensor digitorum longus) and a slow-twitch one (soleus) of the normal adult rat. Because no single measure can describe precisely all geometrical properties (ie both topology and metrics) of the nerve terminals, we evaluated morphologic parameters defining length and angular characteristics in the different terminal segments classified according to their centrifugal order. The main results indicate that the distal free-end segments in the extensor digitorum longus muscle are shorter and less divergent than in the soleus nerve terminals. The endings in the two muscles have different fractal dimensions. Findings are discussed in the context of the hypothetical mechanisms governing motor nerve terminal size and complexity.     

Activity-dependent plastic changes in the motor nerve terminals of the adult rat

Summary— Small and short-lasting physiologic variations in the locomotor activity of normal adult rats can induce remodelling in the motor nerve endings of the fast extensor digitorum longus muscle. The specificity and relative importance of the different plastic adaptations occurring in the presynaptic axonal tree have been studied, in silver impregnated nerve endings, by using an automatic image analysis treatment of the nerve terminals' geometric properties and a discriminant analysis of the morphometric parameters. Changes observed, like selective length variations in certain terminal segments and positional rearrangements, agree with a mechanism of neural connectivity regulation in the adult that arises as a consequence of normal neuromuscular activity.     

Neural regulation of acetylcholinesterase-associated collagen Q in rat skeletal muscles

Acetylcholinesterase-associated collagen Q is expressed also outside of neuromuscular junctions in the slow soleus muscle, but not in fast muscles. We examined the nerve dependence of muscle collagen Q expression and mechanisms responsible for these differences. Denervation decreased extrajunctional collagen Q mRNA levels in the soleus muscles and junctional levels in fast sternomastoid muscles to about one third. Cross-innervation of denervated soleus muscles by a fast muscle nerve, or electrical stimulation by 'fast' impulse pattern, reduced their extrajunctional collagen Q mRNA levels by 70–80%. In contrast, stimulation of fast muscles by 'slow' impulse pattern had no effect on collagen Q expression. Calcineurin inhibitors tacrolimus and cyclosporin A decreased collagen Q mRNA levels in the soleus muscles to about 35%, but did not affect collagen Q expression in denervated soleus muscles or the junctional expression in fast muscles. Therefore, high extrajunctional expression of collagen Q in the soleus muscle is maintained by its tonic nerve-induced activation pattern via the activated Ca²⁺-calcineurin signaling pathway. The extrajunctional collagen Q expression in fast muscles is independent of muscle activation pattern and seems irreversibly suppressed. The junctional expression of collagen Q in fast muscles is partly nerve-dependent, but does not encompass the Ca²⁺-calcineurin signaling pathway.     

Role of excitatory amino acids in the direct and indirect presynaptic regulation of dopamine release from nerve terminals of nigrostriatal dopaminergic neurons

Summary In vivo experiments carried out in halothane-anaesthetized cats implanted with push-pull cannulae demonstrated that glutamate (GLU) released from corticostriatal fibers triggers the release of dopamine (DA), even in the absence of activity in nigral DA cells. As shown in vitro, using rat striatal slices or synaptosomes or in vivo in the cat, both NMDA and AMPA receptors subtypes are involved in the GLU-induced release of DA. Beside this direct regulation, GLU also exert several indirect facilitatory and inhibitory controls on DA release, particularly through cholinergic and GABAergic striatal neurons. Indeed, as shown by numerous authors, the GLU-evoked release of DA is markedly reduced in the presence of tetrodotoxin, bicuculline or atropine or by previous kainate- or ibotenate-induced lesion of striatum. Differences in the presynaptic regulation of DA release in striosomal and matrix compartments have also been found with NMDA and acetylcholine. The effect of acetylcholine was of shorter duration in the matrix than in the striosomal-enriched areas. Two opposite indirect regulations of DA release could be demonstrated: one is facilitatory and involves nicotinic receptors, the other is inhibitory, involves muscarinic receptors and mediated, at least in the matrix by dynorphin containing neurons. The NMDA-evoked responses are of larger amplitude and more sensitive to tetrodotoxin in the matrix than in the striosomes. In conclusion, GLU released from corticostriatal fibers, is able to control the release of DA from terminals of nigrostriatal neurons through direct facilitatory mechanisms (NMDA and AMPA receptors), but also through indirect facilitatory and inhibitory local circuits involving cholinergic and GABAergic neurons.     

Superextension and supercontraction in locust ovipositor muscles

A ten times elongation of certain abdominal intersegmental muscles occurs in female locusts during digging prior to oviposition. During and after oviposition the muscles contract, shortening by up to 90% or more, restoring the resting positions of the abdominal segments.Discontinuous Z-discs permit supercontraction at the resting length and then fragment into Z-bodies when the muscle is stretched, so enabling it to superextend without loss of the contractile property. In this superextended state the fibres resemble smooth muscles. After oviposition, the muscle fibres contract but the sarcomeres are not restored completely, some of the Z-bodies being unevenly distributed in the recontracted fibres. Locust ovipositor muscle has the most extreme example of Z-disc disagregation known from the insects and is the insect muscle which approaches most closely the smooth muscle condition.Two types of motor nerve innervate this muscle, one is ordinary and the other, containing granules, resembles an octopaminergic fibre possibly involved in regulating a catch mechanism in the muscle.The physiological requirements for egg-laying with an extensible ovipositor, which is also part of the normally functioning abdomen, are well met by the ultrastructural specializations of locust ovipositor muscles.     

Adenylate cyclase-mediated modulation of interaction between excitatory and inhibitory synaptic influences on smooth muscles

We found that nonadrenergic inhibitory synaptic potentials (ISP) induced by intramural stimulation in atropine-treated smooth muscles of the guinea-pig large intestine demonstrated no changes upon the influence of an activator of adenylate cyclase, forskolin. This indicates that cAMP-dependent pathways are not involved in the generation of ISP. However, in these muscles with no atropine pretreatment ISP were suppressed by forskolin; intramural stimulation evoked in these smooth muscle cells M-cholinergic excitatory synaptic potentials (ESP) instead of ISP. An increase in the intracellular cAMP concentration due to application of its membrane-penetrating form, dibutyryl-cAMP, did not mimic the above-described effect of forskolin. Hence, it can be supposed that the effect of forskolin on inhibitory synaptic transmission in the atropine-untreated smooth muscles is not related to changes in the intracellular cAMP level; this effect is determined by other mechanisms. The above differences between the effects of forskolin on ISP in the atropine-treated and atropine-untreated smooth muscle strips indicate that the interaction of intracellular signal pathways (probably, through protein G_q/11), which is observed with activation of adenylate cyclase, occurs under conditions of simultaneous activation of M cholinoreceptors and purinoreceptors. The pattern of adenylate cyclase-mediated modulation of inhibitory effects of purinergic neurons on smooth muscles does not allow us to rule out the possibility of involvement of interstitial cells of Cajal as a relay link providing this synaptic effect. Transmission of excitation from cholinergic nerve terminals to smooth muscles is realized without the participation of the interstitial cells of Cajal.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 438–445, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

Interaction between the Cholinergic and Purinergic Control of Transmitter Release in the Neuromuscular Junction

The interaction between the cholinergic and purinergic receptors in the frog neuromuscular junction was studied using a standard microelectrode technique. The inhibitory action of an acetylcholine analog, carbachol, on transmitter release virtually disappeared when the releasing machinery was initially blocked by adenosine, indicating the existence of a functional cross-talk between the purinergic and cholinergic receptors.     

Testosterone control of endplate and non-endplate acetylcholinesterase in the rat levator ani muscle

The time course of effects of castration (5–60 days) and testosterone treatment (15–60 days) of adult male rats were examined on the endplate (+EP) and non-endplate (–EP) acetylcholinesterase (AChE) of the androgen-dependent levator ani (LA) muscle. The thiocholine method was used to determine the enzyme activity. Castration caused LA muscle atrophy within 5 days but reduced the –EP and +EP AChE after 10 and 20 days, respectively. Following 30 days castration –EP and +EP AChE reached respectively 41% and 35% of control activity. Testosterone retrieval restored the control values of both muscle weight and total AChE after 15 and 60 days, respectively. Recovery of the +EP AChE preceded that of –EP AChE by 30 days. The results showed that in the rat LA muscle, +EP and –EP AChE depend on a continuous testosterone regulation that predominates at +EP region spreading thereafter to –EP region. Those data suggest a hormone regulation of AChE exerted indirectly through the synthesis and release of neurotrophic substances.     

Defective peripheral nerve myelination and neuromuscular junction formation in fukutin-deficient chimeric mice

Dystroglycan is a central component of the dystrophin-glycoprotein complex that links the extracellular matrix with cytoskeleton. Recently, mutations of the genes encoding putative glycosyltransferases were identified in several forms of congenital muscular dystrophies accompanied by brain anomalies and eye abnormalities, and aberrant glycosylation of alpha-dystroglycan has been implicated in their pathogeneses. These diseases are now collectively called alpha-dystroglycanopathy. In this study, we demonstrate that peripheral nerve myelination is defective in the fukutin-deficient chimeric mice, a mouse model of Fukuyama-type congenital muscular dystrophy, which is the most common alpha-dystroglycanopathy in Japan. In the peripheral nerve of these mice, the density of myelinated nerve fibers was significantly decreased and clusters of abnormally large non-myelinated axons were ensheathed by a single Schwann cell, indicating a defect of the radial sorting mechanism. The sugar chain moiety and laminin-binding activity of alpha-dystroglycan were severely reduced, while the expression of beta1-integrin was not altered in the peripheral nerve of the chimeric mice. We also show that the clustering of acetylcholine receptor is defective and neuromuscular junctions are fragmented in appearance in these mice. Expression of agrin and laminin as well as the binding activity of alpha-dystroglycan to these ligands was severely reduced at the neuromuscular junction. These results demonstrate that fukutin plays crucial roles in the myelination of peripheral nerve and formation of neuromuscular junction. They also suggest that defective glycosylation of alpha-dystroglycan may play a role in the impairment of these processes in the deficiency of fukutin.     

Structure and function of the spermathecal complex in the phlebotomine sandflyPhlebotomus papatasi Scopoli (Diptera: Psychodidae): I. Ultrastructure and histology

Females of phlebotomine sandflies (Diptera: Psychodidae) possess highly variable spermathecae that present several important taxonomic characters. The cause of this diversity remains a neglected field of sandfly biology, but may possibly be due to female post-mating sexual selection. To understand this diversity, a detailed study of the structure and function of the spermathecal complex in at least one of the species was a prerequisite. Using scanning and transmission electron microscopy, described here is ultrastructure of the spermathecal complex in the sand fly,Phlebotomus papatasi Scopoli. The spermathecal complexes are paired; each consists of a long spermathecal duct, a cylindrical spermathecal body, and a spherical spermathecal gland. Muscle fibres, nerves, tracheoles, and vascular sinuses connect the spermathecal body and duct through the epithelial layers. Spermathecal gland is formed by a typical insect epidermis and consisting of an epithelial layer of class-1 epidermal cells and elaborate glandular cells of class-3 epidermal cells, each having both receiving and conducting ductules (i.e. “end apparatus”) and a “cytological apodeme”, which is a newly described cell structure. The spermathecal body and duct are lined by class-1 epidermal cells and a cuticle, and are enveloped by a super-contracting visceral muscular system. The cuticle consists of rubber-like resilin, and its fibrillar arrangement and chemical nature are described. A well-developed neuromuscular junction exists between the spermathecal gland and the spermathecal body, which are connected to each other by a nerve and a muscle. The spermathecal complexes of the sandfly are compared with those of other insect species. The physiological role and possible evolutionary significance of the different parts of spermathecal complex in the sandfly are inferred from the morphology and behaviour. Post-mating sexual selection may be responsible for the structural uniqueness of the spermathecal complex in phlebotomine sandflies.     

Transient and permanent effects of androgen during synapse elimination in the levator ani muscle of the rat

Young male rats were castrated at 7 days of age, and treated with testosterone propionate daily from 7 to 34 days of age. At 13 months of age, motor axons and terminals innervating the levator ani (LA) muscle were stained with tetranitroblue tetrazolium (TNBT). The number of separate axons innervating individual muscle fibers was counted, and muscle fiber diameter was measured. Previous studies have shown that this androgen treatment increases muscle fiber diameter and delays synapse elimination, measured as (1) a greater percentage of muscle fibers innervated by multiple axons and (2) larger motor units. The present results indicate that the androgenic effect on synapse elimination is permanent, in that high levels of multiple innervation persisted for 12 months after the end of androgen treatment. In contrast, the effect on muscle fiber diameter was not maintained for this period. This dissociation of androgenic effects on the pattern of innervation from androgenic effects on muscle fiber diameter offers further evidence that the androgenic maintenance of multiple innervation is not dependent on muscle fiber size. In addition, circulating testosterone levels were measured at 50 and 60 days of age in animals similarly treated with androgen or oil from 7 to 34 days of age. By 60 days of age, testosterone levels in hormone-treated animals had dropped below detectability, comparable to levels in oil-treated controls. This provides additional evidence that androgen treatment during juvenile development can have permanent effects on the adult pattern of innervation in the LA muscle.     

Peculiarities of synaptic vesicle recycling in frog and mouse motor nerve terminals

Using electrophysiology and fluorescence microscopy with dye FM 1-43, a comparative study of peculiarities of neurotransmitter secretion, synaptic vesicle exo-endocytosis and recycling has been carried out in nerve terminals (NT) of the skin-sternal muscle of the frog Rana ridibunda and of the white mouse diaphragm muscle during a long-term high-frequency stimulation (20 imp/s). The obtained data have allowed identifying three synaptic vesicle pools and two recycling ways in the motor NT. In the frog NT, the long-term high-frequency stimulation induced consecutive expenditure of the pool ready to release, the mobilizational, and reserve vesicle pools. The exocytosis rate exceeded markedly the endocytosis rate; the slow synaptic vesicle recycling with replenishment of the reserve pool was predominant. In the mouse NT, only the vesicles of the ready to release and the mobilizational pools, which are replenished predominantly by fast recycling, were exocytosed. The exo- and endocytosis occurred practically in parallel, while vesicles of the reserve pool did not participate in the neurotransmitter secretion. It is suggested that evolution of the motor NT from the poikilothermal to homoiothermal animals went by the way of a decrease of the vesicle pool size, the more economic expenditure and the more effective reuse of synaptic vesicles owing to the high rates of endocytosis and recycling. These peculiarities can provide in NT of homoiothermal animals a long maintenance of neurotransmitter secretion at the steady and sufficiently high level to preserve reliability of synaptic transmission in the process of the high-frequency activity.     

Effect of calcitonin gene-related peptide on the neuroeffector mechanism of sympathetic nerve terminals in rat vas deferens

In order to evaluate the mode of action of calcitonin gene-related peptide (CGRP) on the neuroeffector mechanism of peripheral sympathetic nerve fibers, the effects of CGRP were tested on the electrical stimulated and the non-stimulated preparations of the isolated rat vas deferens. The contractile responses, which were mediated predominantly by activation of postganglionic noradrenergic nerve fibers, were dose-dependently inhibited by CGRP in concentrations ranging from 0.1 to 10 nM. The inhibitory response produced by CGRP in high concentrations (greater than 2 nM) usually returned to the control level at 20-30 min and were rarely tachyphylactic. The inhibitory action of CGRP was not modified by pretreatment with 10(-7) M propranolol or 10(-7) M atropine. Contractions produced by exogenous norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in unstimulated preparations were not affected by pretreatment with CGRP in a low concentration (less than 2 nM). On the other hand, the contractions were slightly reduced 1 min after pretreatment with CGRP in high concentrations (greater than 5 nM), which recovered in 15 min after constant flow washout. High concentrations of CGRP also caused a concentration-dependent relaxation on the precontracted preparations produced by high potassium (60 mM K+) solution. These results suggest that CGRP in high concentrations (greater than 5 nM) may have a non-specific inhibitory action on the postsynaptic plasma membrane of the smooth muscle cell and a postulated CGRP receptor exists presynaptically in the rat vas deferens and that CGRP may inhibit the release of NE during adrenergic nerve stimulation.     

Shared and unique roles of CAP23 and GAP43 in actin regulation, neurite outgrowth, and anatomical plasticity

CAP23 is a major cortical cytoskeleton-associated and calmodulin binding protein that is widely and abundantly expressed during development, maintained in selected brain structures in the adult, and reinduced during nerve regeneration. Overexpression of CAP23 in adult neurons of transgenic mice promotes nerve sprouting, but the role of this protein in process outgrowth was not clear. Here, we show that CAP23 is functionally related to GAP43, and plays a critical role to regulate nerve sprouting and the actin cytoskeleton. Knockout mice lacking CAP23 exhibited a pronounced and complex phenotype, including a defect to produce stimulus-induced nerve sprouting at the adult neuromuscular junction. This sprouting deficit was rescued by transgenic overexpression of either CAP23 or GAP43 in adult motoneurons. Knockin mice expressing GAP43 instead of CAP23 were essentially normal, indicating that, although these proteins do not share homologous sequences, GAP43 can functionally substitute for CAP23 in vivo. Cultured sensory neurons lacking CAP23 exhibited striking alterations in neurite outgrowth that were phenocopied by low doses of cytochalasin D. A detailed analysis of such cultures revealed common and unique functions of CAP23 and GAP43 on the actin cytoskeleton and neurite outgrowth. The results provide compelling experimental evidence for the notion that CAP23 and GAP43 are functionally related intrinsic determinants of anatomical plasticity, and suggest that these proteins function by locally promoting subplasmalemmal actin cytoskeleton accumulation.     

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.     

Exercise-dependent regulation of glial cell line-derived neurotrophic factor (GDNF) expression in skeletal muscle and its importance for the neuromuscular system

The focus of this review is to highlight the importance of glial cell line-derived neurotrophic factor (GDNF) for the motor nervous system. GDNF is the most potent survival factor for motor neurons, where it enhances maintenance and survival of both developing and mature motor neurons in vivo and in vitro. GDNF aids in neuromuscular junction formation, maintenance, and plasticity, where skeletal muscle-derived GDNF may be responsible for this phenomenon. Increased levels of physical activity can increase GDNF protein levels in skeletal muscle, where alterations in acetylcholine and acetylcholine receptor activation may be involved in regulation of these changes observed. With inactivity and disuse, GDNF expression shows different patterns of regulation in the central and peripheral nervous systems. Due to its potent effects for motor neurons, GDNF is being extensively studied in neuromuscular diseases.     

Ultrastructural relation between cortical efferents and terminals containing enkephalin-like immunoreactivity in rat neostriatum

The interrelationships between cortical efferents and terminals containing enkephalin-like immunoreactivity (ELI) were examined by combining anterograde degeneration with electron microscopic immunocytochemistry in the adult rat neostriatum. Two days following unilateral removal of the cerebral cortex, the brains were fixed by aortic arch perfusion, then sectioned and processed for the immunocytochemical localization of an antiserum directed against methionine (Met5)-enkephalin. The observed relationships between the degenerating cortical efferents and immunocytochemically labeled terminals were of two types. In the first, the degenerating and ELI containing terminals converged on the same unlabeled dendrite or dendritic spine. In the second, terminal and preterminal axons of the ELI containing neurons had one surface directly apposed to the plasma membrane of a degenerating axon terminal. These findings support the concept that neurons containing opioid peptides and cortical efferents modulate the output of common recipient neurons and may also directly interact with each other through presynaptic axonal mechanisms in the rat neostriatum.     

Similarities and differences between frozen-hydrated, rigor acto-S1 complexes of insect flight and chicken skeletal muscles

The structure and function of myosin crossbridges in asynchronous insect flight muscle (IFM) have been elucidated in situ using multiple approaches. These include generating “atomic” models of myosin in multiple contractile states by rebuilding the crystal structure of chicken subfragment 1 (S1) to fit IFM crossbridges in lower-resolution electron microscopy tomograms and by “mapping” the functional effects of genetically substituted, isoform-specific domains, including the converter domain, in chimeric IFM myosin to sequences in the crystal structure of chicken S1.We prepared helical reconstructions (∼ 25 Å resolution) to compare the structural characteristics of nucleotide-free myosin0 S1 bound to actin (acto-S1) isolated from chicken skeletal muscle (CSk) and the flight muscles of Lethocerus (Leth) wild-type Drosophila (wt Dros) and a Drosophila chimera (IFI-EC) wherein the converter domain of the indirect flight muscle myosin isoform has been replaced by the embryonic skeletal myosin converter domain. Superimposition of the maps of the frozen-hydrated acto-S1 complexes shows that differences between CSk and IFM S1 are limited to the azimuthal curvature of the lever arm: the regulatory light-chain (RLC) region of chicken skeletal S1 bends clockwise (as seen from the pointed end of actin) while those of IFM S1 project in a straight radial direction. All the IFM S1s are essentially identical other than some variation in the azimuthal spread of density in the RLC region. This spread is most pronounced in the IFI-EC S1, consistent with proposals that the embryonic converter domain increases the compliance of the IFM lever arm affecting the function of the myosin motor. These are the first unconstrained models of IFM S1 bound to actin and the first direct comparison of the vertebrate and invertebrate skeletal myosin II classes, the latter for which, data on the structure of discrete acto-S1 complexes, are not readily available.