A central role for denervated tissues in causing nerve sprouting

One of the oldest known forms of neuronal plasticity is the ability of peripheral nerves to grow and form functional connections after damage to neighbouring axons. Yet the source of the signal which elicits this "sprouting" remains unknown. In mammalian muscles, paralysis-which gives rise to many of the changes which occur in denervated muscles-causes motor nerve terminals to sprout. Could the inactive muscle fibres (rather than nerve degeneration products, another likely source) be responsible for some of the sprouting found in partial denervation? We confirm in this paper that direct stimulation of a partially denervated muscle inhibits sprouting and show that stimulation does so by activating the denervated fibres. Consequently after partial denervation the same signal as that which causes terminal sprouting in a paralysed muscle is able to spread from the denervated muscle fibres to the nerves on the innervated fibres and initiate terminal sprouting.     

Suppression of sprouting at the neuromuscular junction by immune sera

Injury of afferent motor axons or pathological loss of motoneurones from the spinal cord causes the remaining axons within a muscle to sprout and to reinnervate the denervated muscle fibres. Sprouting occurs at two sites along intramuscular axons, at nodes of Ranvier (nodal sprouting) and at the neuromuscular junction (terminal sprouting). Terminal sprouting is also produced by treatment with botulinum toxin and by other agents that render muscle inactive. The muscle probably provides a signal for terminal sprouting as restoration of muscle activity by direct electrical stimulation prevents sprouting. Such a signal might be a local change on the muscle fibre surface or a 'soluble' sprouting factor, although the failure to induce terminal sprouting in one muscle by denervating adjacent muscles argues against the latter hypothesis. I now report that rabbit antisera against a 56,000 (56K)-molecular weight protein secreted by denervated rat muscle suppress botulinum toxin-induced terminal sprouting in the mouse gluteus muscle. An immune response against this protein has also been detected in serum of patients with amyotrophic lateral sclerosis (ALS), a disease in which loss of motoneurones from the spinal cord is not accompanied by the degree of sprouting and reinnervation seen in other motoneurone diseases.     

Local regulation of compensatory noradrenergic hyperactivity in the partially denervated hippocampus

Functional recovery after denervating lesions in the central nervous system (CNS) is particularly prominent if part of the lesioned projection is spared. Several plasticity mechanisms, such as collateral sprouting, hyperactivity of remaining axons and development of receptor supersensitivity, probably contribute to efficient recovery after subtotal lesions. Although denervation-induced collateral sprouting and presynaptic compensatory hyperactivity in spared axons have been described in various systems, any possible interaction or cooperation between the two mechanisms in restoring synaptic transmission in a partially denervated target has so far not been demonstrated. We have shown previously that partial adrenergic denervation of the hippocampus in adult rats is followed by a slow and protracted reinnervation by collateral sprouting from the spared adrenergic afferents. We now report that the partial adrenergic deafferentation is accompanied by a transient increase in turnover of the transmitter in remaining axons which subsides when the denervated region becomes reinnervated, and that the development of this compensatory hyperactivity is confined to the area of maximal denervation. The topographical specificity of the compensatory noradrenergic hyperactivity response, and the interaction between this hyperactivity and the collateral reinnervation process, strongly suggest that the changes in transmitter turnover in spared afferents after denervating lesions can be regulated by local mechanisms operating within the denervated target area.     

A physiological correlate of disuse-induced sprouting at the neuromuscular junction.

Recent investigations have established that many of the normal properties of muscle fibres are maintained, at least in part, by muscle activity. Thus, a fall in resting membrane potential, an increase in input resistance, and spread of acetylcholine receptors to extrajunctional sites can all be induced by abolishing muscle activity and prevented by direct stimulation of denervated muscle fibres. Muscle activity also exerts a trophic influence on the innervating motoneurones; furthermore it may be a factor in the regulation of sprouting. Brown and Ironton found fine, "ultra-terminal sprouts" emanating from the endplates of muscles rendered inactive by chronic conduction block of the muscle nerve. Pestronk and Drachman saw increased branching of the motor nerve terminal and a consequent increase in endplate size in similar conditions. If these sprouts at the endplates of inactive muscles were functional, one might expect more transmitter to be released in response to nerve stimulation. We report here that both quantum content and spontaneous miniature endplate potential (m.e.p.p) frequency are increased at the terminals of inactive (disused) muscles.     

Common mechanisms of nerve and blood vessel wiring

Blood vessels and nerve fibres course throughout the body in an orderly pattern, often alongside one another. Although superficially distinct, the mechanisms involved in wiring neural and vascular networks seem to share some deep similarities. The discovery of key axon guidance molecules over the past decade has shown that axons are guided to their targets by finely tuned codes of attractive and repulsive cues, and recent studies reveal that these cues also help blood vessels to navigate to their targets. Parallels have also emerged between the actions of growth factors that direct angiogenic sprouting and those that regulate axon terminal arborization.     

Long-term regenerated nerve fibres retain sensitivity to potassium channel blocking agents

Mammalian myelinated peripheral nerve fibres display a remarkable degree of regeneration following a discrete nerve crush. Nerve crush disrupts the axon cylinder, but leaves the basement membrane of the Schwann cell intact. These intact endoneurial tubes provide pathways to guide the regenerating axon sprouts. After contact with the periphery is established, the regenerating fibres enlarge and myelinate. Conduction velocity recovers to nearly normal and functional recovery is, in many cases, nearly complete. A distinct feature of normal mature myelinated axons is the insensitivity of these fibres to potassium channel blocking agents. In contrast, immature myelinated axons are exquisitely sensitive to the K channel blocking agent 4-aminopyridine (4-AP). Application of 4-AP to immature myelinated fibres leads to a delayed membrane depolarization with action potential burst activity in response to a single stimulus. This sensitivity to 4-AP is attenuated as the fibres mature. Previous studies have demonstrated a sensitivity to 4-AP in regenerating nerve fibres; this sensitivity differentiates the regenerating axon segments from their normal parent axon segments. Such studies have not, however, examined the question of whether regenerated fibres, which have re-established peripheral connections and are functionally active, fully recapitulate the functional organization of normal mature myelinated fibres. We demonstrate here that while sensitivity to the potassium channel blocking agents 4-AP and 3, 4-diaminopyridine (3, 4-DAP) is lost in the normal course of myelinated axon maturation, this property is present in long-term regenerated axons. This suggests that long-term regenerated mammalian axons are characterized by a functional organization that bears a closer resemblance to that of immature myelinated fibres than to that of adult myelinated fibres.     

Axoplasmic transport of muscarinic receptors

The reality of axoplasmic transport is widely accepted; various neutrotransmitters, enzymes, labelled proteins and peptides are known to move rapidly along the axons of different nerve fibres. In the terminals of sympathetic nerves, noradrenaline release is controlled by various regulatory mechanisms which imply the occurrence of presynaptic receptors. In this regard, there is considerable indirect physiological evidence for the existence of muscarinic cholinergic receptors in the sympathetic nerve endings; the stimulation by acetylcholine of such presynaptic receptors elicits an inhibitory effect on noradrenaline release. We not provide direct biochemical evidence for the occurrence in dog splenic nerve of muscarinic receptors which seem to move along the axon as suggested by their rapid accumulation on either side of a ligature.     

Synapse elimination in neonatal rat muscle is sensitive to pattern of muscle use

The synaptic connections among the cells of the vertebrate nervous system undergo extensive rearrangements early in development. During their initial growth, neurones apparently form synaptic connections with an excessive number of targets, later retracting a portion of these synapses in establishing the adult neural circuits. Because of the profound effects which experience has upon the developing nervous system, a question of considerable interest has been the role which the functional use of these developing synapses might play in determining the final pattern of connectivity. At the neuromuscular junction the early changes in synaptic connections are well documented, and here questions about the importance of function can be relatively easily addressed. Mammalian skeletal muscle fibres experience a perinatal period of synapse elimination so that all but one of several synapses formed on each muscle fibre are lost. This synapse elimination is sensitive to alterations of neuromuscular use or activity. Reduction of muscle use by tenotomy or by paralysis of the muscle with drugs blocking nerve impulse conduction or neuromuscular transmission delays or even prevents synapse loss, while increased use produced by stimulation of the muscle nerve apparently accelerates the rate at which synapses are lost. I report here a further examination of the role of neuromuscular activity in synapse elimination. I show that chronic neuromuscular stimulation accelerates synapse elimination but that this acceleration is dependent on the temporal pattern in which the stimuli are presented: brief stimulus trains containing 100 Hz bursts of stimuli produce this acceleration whereas the same number of stimuli presented continuously at 1 Hz do not. Furthermore, the 100 Hz activity pattern which is effective in altering synapse elimination also alters two other muscle properties: the sensitivity of the muscle fibers to acetylcholine and the 'speed' of muscle contractions. These findings suggest that the ability of muscle fibres to maintain more than one nerve terminal, like other muscle properties, is sensitive to the pattern of muscle use rather than just the total amount of use.     

Retinofugal fibres change conduction velocity and diameter between the optic nerve and tract in ferrets

In earlier studies of central nervous fibre tracts, it was tacitly assumed that individual axons are relatively uniform along their length. In the retinofugal pathway in particular, axon diameter, myelin thickness and correlated conduction properties have been treated as constant throughout the optic nerve, chiasm and tract. We report here that the conduction velocities of fibres contributing to the early components of the compound action potential are significantly greater in the optic tract than in the optic nerve of ferrets, and also that the diameters of the largest retinofugal fibres increase from nerve to tract. This observation raises significant questions about the developmental mechanisms in the central nervous system that relate the axons, their diameters, and the glia with which they are myelinated. In addition, it indicates that studies that have relied on the constancy of conduction velocity along the retinofugal course may require reappraisal.     

Dystrophic mice show age related muscle fibre and myelinated axon losses

Although many differences between age matched normal and dystrophic animals have been found, there have been few demonstrations of a time related quantitative change from normal to a characteristically dystrophic situation within the dystrophic strain itself. Here we report such a change-normal numbers of muscle fibres present in young dystrophic mice were rapidly lost, such that older animals showed the reduced number of muscle fibres characteristic of murine dystrophy. Although these losses began after a demonstrable loss of myelinated axons had occurred, it is not possible to say if the loss of muscle fibres was a result of the loss of nerve fibres.     

Evidence for neurotensin as a non-adrenergic, non-cholinergic neurotransmitter in guinea pig ileum

Neurotensin is a 13-amino acid peptide that is widely distributed in central and peripheral tissues of various mammalian species. In peripheral tissues, the highest concentration of neurotensin-like immunoreactivity is found in the ileum, where it is present in endocrine-like cells and nerve fibres. The longitudinal smooth muscle layer of the guinea pig ileum, where neurotensin has both a direct relaxant and an indirect contractile action, has been used extensively as a biological assay system for neurotensin. We report here that the majority of specific 3H-neurotensin binding sites is present in the guinea pig ileum circular smooth muscle layer, which is known to be innervated by a large proportion of the ileal non-adrenergic inhibitory nerves. Neurotensin produces a dose-dependent, tetrodotoxin-resistant relaxation, whereas the relaxation produced by field stimulation of the inhibitory nerves is frequency-dependent and tetrodotoxin-sensitive. The calcium-dependent potassium channel blocker apamin inhibits both the neurotensin- and nerve stimulation-induced muscle relaxation. Incubation of the circular smooth muscle preparation with a neurotensin antiserum substantially inhibited the nerve stimulation-induced relaxation, indicating a direct relationship between the effects of neurotensin and of nerve stimulation.     

Modification of retinal ganglion cell axon morphology by prenatal infusion of tetrodotoxin

The cellular mechanisms by which the axons of individual neurons achieve their precise terminal branching patterns are poorly understood. In the visual system of adult cats, retinal ganglion cell axons from each eye form narrow cylindrical terminal arborizations restricted to alternate non-overlapping layers within the lateral geniculate nucleus (LGN). During prenatal development, axon arborizations from the two eyes are initially simple in shape and are intermixed with each other; they then gradually segregate to form complex adult-like arborizations in separate eye-specific layers by birth. Here we report that ganglion cell axons exposed to tetrodotoxin (TTX) to block neuronal activity during fetal life fail to form the normal pattern of terminal arborization. Individual TTX-treated axon arborizations are not stunted in their growth, but instead produce abnormally widespread terminal arborizations which extend across the equivalent of approximately two eye-specific layers. These observations suggest that during fetal development of the central nervous system, the formation of morphologically appropriate and correctly located axon terminal arborizations within targets is brought about by an activity-dependent process.     

LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite

Structural remodelling of synapses and formation of new synaptic contacts has been postulated as a possible mechanism underlying the late phase of long-term potentiation (LTP), a form of plasticity which is involved in learning and memory. Here we use electron microscopy to analyse the morphology of synapses activated by high-frequency stimulation and identified by accumulated calcium in dendritic spines. LTP induction resulted in a sequence of morphological changes consisting of a transient remodelling of the postsynaptic membrane followed by a marked increase in the proportion of axon terminals contacting two or more dendritic spines. Three-dimensional reconstruction revealed that these spines arose from the same dendrite. As pharmacological blockade of LTP prevented these morphological changes, we conclude that LTP is associated with the formation of new, mature and probably functional synapses contacting the same presynaptic terminal and thereby duplicating activated synapses.     

Changes in spinal cord reflexes after cross-anastomosis of cutaneous and muscle nerves in the adult rat

Evidence exists that the specification of afferent nerves and their central connections in the embryo may depend in part on influences from the peripheral target innervated. We have now investigated whether such peripheral determination persists in the adult rat using the unmyelinated afferent system of C fibres, which differ chemically in the adult depending on their target. We have previously shown that if the cutaneous sural nerve and the muscle gastrocnemius nerve are cross-anastomosed so that they grow to each other's target, the A fibres establish functional endings and the C fibres change their chemistry to that which is appropriate for the new target. Here we report that in normal adult rats, a short train of stimuli to the cutaneous sural nerve produced a brief facilitation of the flexion reflex, lasting on average only 5 min, whereas similar stimulation of the gastrocnemius-muscle nerve enhanced this reflex for an average of 54 min. In cross-anastomosed animals, stimulation of the gastrocnemius nerve (innervating skin) induced a brief potentiation of the flexion reflex, lasting on average only 3 min. By contrast, stimulation of sural nerve (innervating muscle) produced a potentiation of this reflex lasting 57 min. Thus the ability of adult afferent nerves to potentiate the flexion reflex depends on the target with which they make contact. We propose that tissue-specific factors influence some of the central actions of primary afferent neurons in the adult.     

Potassium channels in the nodal membrane of rat myelinated fibres

Following some preliminary reports, mammalian fibres from rabbit and rat have recently been successfully studied in detail by means of the voltage clamp. The early transient or sodium conductance system was found to be similar to that in frog and squid axons. However, the delayed conductance or potassium currents were found to be negligible. Only after chemical and osmotic manipulations, which were said to expose channels buried under the myelin, did Chiu and Ritchie find delayed currents in rabbit fibres. If confirmed, this would mean that the membrane conductance system of mammalian fibres is so different from that of invertebrate and amphibian axon models as to make the data base gathered from amphibian myelinated fibres (frog and toad) and invertebrate giant axons (squid and myxicola) irrelevant to human nd other mammalian fibres. However, we show here that it is possible to find in the normal nodal membrane of rat myelinated fibres potassium currents that flow through channels which are similar in many respects to those found in the frog node of squid axons.     

Oligodendroglia metabolically support axons and contribute to neurodegeneration

Oligodendroglia support axon survival and function through mechanisms independent of myelination, and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been proposed. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and use. Here we show that the most abundant lactate transporter in the central nervous system, monocarboxylate transporter 1 (MCT1, also known as SLC16A1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and in mouse models of, amyotrophic lateral sclerosis, suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.     

Differential localization of type I and type II benzodiazepine binding sites in substantia nigra

A number of studies have suggested the existence of multiple benzodiazepine binding sites in the brain. We have recently reported the physical separation of two apparent benzodiazepine binding site subtypes, the pharmacological properties, and distribution in tissue sections of which correspond to the putative type I and type II sites. Benzodiazepine and gamma-aminobutyric acid (GABA) receptors have been shown to interact, and lesions of the GABAergic striatonigral pathway, which lead to GABA supersensitivity, both increase the numbers of GABA binding sites and enhance GABA-stimulated benzodiazepine binding. We demonstrate here that degeneration of striatonigral fibres increases the density of putative type I benzodiazepine binding sites in the substantia nigra and decreases the density of the putative type II sites. This suggests that type I sites that increase after denervation are postsynaptic, whereas the type II sites reduced by the lesion may be localized to axons or terminals of the striatonigral pathways.     

Fluorescently labelled Na+ channels are localized and immobilized to synapses of innervated muscle fibres

Segregation of voltage-dependent sodium channels to the hillock of motoneurones and nodes of Ranvier in myelinated axons is crucial for conduction of the nerve impulse. Much less is known, however, about the distribution of voltage-dependent Na+ channels on muscle fibres. Recently, Beam et al. have shown that Na+ channels are concentrated near the neuromuscular junction. To determine the topography and mechanisms governing the distribution of voltage-dependent Na+ channels on muscle, microfluorimetry and fluorescence photobleach recovery (FPR) have now been used to measure the density and lateral mobility of fluorescently labelled Na+ channels on uninnervated and innervated muscle fibres. On uninnervated myotubes, Na+ channels are diffusely distributed and freely mobile, whereas after innervation the channels concentrate at neuronal contact sites. These channels are immobile and co-localize with acetylcholine receptors (AChRs). At extrajunctional regions the Na+ channel density is lower and the channels more mobile. The results suggest that the nerve induces Na+ channels to redistribute, immobilize and co-localize with AChRs at sites of neuronal contact.     

Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses

IN mammalian muscle, the subunit composition of the nicotinic acetylcholine receptor (AChR) and the distribution of AChRs along the fibre are developmentally regulated. In fetal muscle, AChRs are distributed over the entire fibre length whereas in adult fibres they are concentrated at the end-plate. We have used in situ hybridization techniques to measure the development of the synaptic localization of the messenger RNAs (mRNAs) encoding the alpha-subunit and the epsilon-subunit of the rat muscle AChR. The alpha-subunit is present in both fetal and adult muscle, whereas the epsilon-subunit appears postnatally and specifies the mature AChR subtype. The synaptic localization of alpha-subunit mRNA in adult fibres may arise from the selective down-regulation of constitutively expressed mRNA from extrasynaptic fibre segments. In contrast, epsilon-subunit mRNA appears locally at the site of neuromuscular contact and its accumulation at the end-plate is not dependent on the continued presence of the nerve terminal very early during synapse formation. This suggests that epsilon-subunit mRNA expression is induced locally via a signal which is restricted to the end-plate region and is dependent on the presence of the nerve only during a short period of early neuromuscular contact. Evidently, several mechanisms operate to confine AChR mRNAs to the adult end-plate region, and the levels of alpha-subunit and epsilon-subunit mRNAs depend on these mechanisms to differing degrees.     

Threshold channels--a novel type of sodium channel in squid giant axon

Sodium channels in nerve and muscle cells are functionally similar across wide phylogenetic boundaries and are usually thought to represent a single, homogeneous population that initiates the action potential at threshold and unerringly transmits it along the surface membrane. In marked contrast, many cell types are known to have several distinct potassium permeability systems. Distinguishable populations of Na channels have been reported in a few cell types, however, including denervated skeletal muscle, embryonic cardiac muscle, Purkinje cell somata and non-myelinated axons at low temperature. We report here that in squid giant axon, in standard experimental conditions, there are two functionally distinct populations of Na channels. The newly discovered population accounts for only a few per cent of the total Na permeability. The channels are selectively activated by small depolarizations and have very slow closing kinetics. Because these channels activate at voltages near the resting potential and tend to stay open for long times, they must dominate behaviour of the axon membrane in the threshold region for action potential initiation.