Specification of synaptic connections between sensory and motor neurons in the developing spinal cord

Experimental studies of mechanisms underlying the specification of synaptic connections in the monosynaptic stretch reflex of frogs and chicks are described. Sensory neurons innervating the triceps brachii muscles of bullfrogs are born throughout the period of sensory neurogenesis and do not appear to be related clonally. Instead, the peripheral targets of these sensory neurons play a major role in determining their central connections with motoneurons. Developing thoracic sensory neurons made to project to novel targets in the forelimb project into the brachial spinal cord, which they normally never do. Moreover, these foreign sensory neurons make monosynaptic excitatory connections with the now functionally appropriate brachial motoneurons. Normal patterns of neuronal activity are not necessary for the formation of specific central connections. Neuromuscular blockade of developing chick embryos with curare during the period of synaptogenesis still results in the formation of correct sensory-motor connections. Competitive interactions among the afferent fibers also do not seem to be important in this process. When the number of sensory neurons projecting to the forelimb is drastically reduced during development, each afferent still makes central connections of the same strength and specificity as normal. These results are discussed with reference to the development of retinal ganglion cells and their projections to the brain. Although many aspects of the two systems are similar, patterned neural activity appears to play a much more important role in the development of the visual pathway than in the spinal reflex pathway described here.     

Ascending and descending projections of the lateral vestibular nucleus in the rat

The tracer neurobiotin was injected into the lateral vestibular nucleus in rat and the efferent fiber connections of the nucleus were studied. The labeled fibers reached the diencephalon rostrally and the sacral segments of the spinal cord caudally. In the diencephalon, the ventral posteromedial and the gustatory nuclei received the most numerous labeled fibers. In the mesencephalon, the inferior colliculus, the interstitial nucleus of Cajal, the nucleus of Darkschewitch, the periaqueductal gray matter and the red nucleus received large numbers of labeled fibers. In the rhombencephalon, commissural and internuclear connections originated from the lateral vestibular nucleus to all other vestibular nuclei. The medioventral (motor) part of the reticular formation was richly supplied, whereas fewer fibers were seen in the lateral (vegetative) part. In the spinal cord, the descending fibers were densely packed in the anterior funiculus and in the ventral part of the lateral funiculus. Collaterals invaded the entire gray matter from lamina IX up to lamina III; the fibers and terminals were most numerous in laminae VII and VIII. Collateral projections were rich in the cervical and lumbosacral segments, whereas they were relatively poor in the thoracic segments of the spinal cord. It was concluded that the fiber projection in the rostral direction was primarily aimed at sensory-motor centers; in the rhombencephalon and spinal cord, fibers projected onto structures subserving various motor functions.     

Formation of functional synaptic connections between heterogeneous brain formations in organotypic nerve tissue culture

The principles of oriented growth of nerve fibers and the formation of functional synaptic connections during combined culture of brain structures with no direct anatomical or functional connections (the spinal cord and olfactory bulbs of mouse embryos) were investigated by neuromorphological and electrophysiological methods. During the first week of culture connections, mainly glio-neuronal bridges, formed between the explants of spinal cord and olfactory bulb. Glial cells forming an oriented substrate, facilitating growth and fasciculation (the formation of bundles) of axons that developed subsequently, play an active role in the formation of such connections. In preparations impregnated with silver, connections formed by bundles of axons or by single nerve fibers were seen between the explants. The results of electrophysiological investigations of combined cultures of heterogeneous brain structures showed that by the second week of culture functional synaptic connections have formed between explants of spinal cord and olfactory bulbs. Electrical stimulation of spinal cord explants led to the appearance of short- and long-latency unit responses in explants of the olfactory bulbs. The formation of nonspecific functional synaptic connections for these brain structures during combined culture, revealed by this investigation, is evidence of the high level of morphogenetic plasticity of growing or regenerating axons and of the active role of neuroglial cells in preparation and provision for oriented growth of nerve fibers.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 12, No. 5, pp. 490–497, September–October, 1980.     

Curare-induced transformation of myosin pattern in developing skeletal muscle fibers

The effects of neuromuscular block on the pattern of distribution of myosin isozymes in developing skeletal muscle fibers was examined by immunocytochemistry. The homogeneous population of fibers in the anterior latissimus dorsi (ALD) of the 18-day chick embryo was converted by curare to a mosaic of at least two categories of fibers. Normally all fibers in this slow muscle reacted with antibodies against slow myosin (anti-ALD). They also reacted with an antibody specific for the alkali 1 light chain (anti-delta 1) but not the alkali 2 light chain (anti-delta 2) of fast myosin. After treatment with curare, which inhibits neuronal cell death and increases the number of axonal endings, ALD muscle fibers continued to react with anti-delta 1, but many now reacted with anti-delta 2 as well. The same fibers failed to react with anti-ALD. From this it can be concluded that the myosin in this population was converted to a type not normally present. The changes, therefore, are not merely a result of the preferential loss of a slow type of fiber, nor are they a result of delayed maturation. In contrast, curare had no apparent effect on the fast posterior latissimus dorsi (PLD). As in the normal muscle at 18 days, all fibers reacted strongly with anti-delta 1 and to variable degrees with anti-delta 2, and very few fibers reacted with anti-ALD. Our observations suggest that the dual response to antibodies against fast and slow myosin during development is not a necessary consequence of multiple axon terminals. We present evidence that curare induces the expression of a different myosin in the embryonic ALD, and we suggest that the selective transformation of the fiber population may be a manifestation of a change in composition of the motoneuron pool.     

Transplantation of Xenopus laevis ears reveals the ability to form afferent and efferent connections with the spinal cord

Previous comparative and developmental studies have suggested that the cholinergic inner ear efferent system derives from developmentally redirected facial branchial motor neurons that innervate the vertebrate ear hair cells instead of striated muscle fibers. Transplantation of Xenopus laevis ears into the path of spinal motor neuron axons could show whether spinal motor neurons could reroute to innervate the hair cells as efferent fibers. Such transplantations could also reveal whether ear development could occur in a novel location including afferent and efferent connections with the spinal cord. Ears from stage 24-26 embryos were transplanted from the head to the trunk and allowed to mature to stage 46. Of 109 transplanted ears, 73 developed with otoconia. The presence of hair cells was confirmed by specific markers and by general histology of the ear, including TEM. Injections of dyes ventral to the spinal cord revealed motor innervation of hair cells. This was confirmed by immunohistochemistry and by electron microscopy structural analysis, suggesting that some motor neurons rerouted to innervate the ear. Also, injection of dyes into the spinal cord labeled vestibular ganglion cells in transplanted ears indicating that these ganglion cells connected to the spinal cord. These nerves ran together with spinal nerves innervating the muscles, suggesting that fasciculation with existing fibers is necessary. Furthermore, ear removal had little effect on development of cranial and lateral line nerves. These results indicate that the ear can develop normally, in terms of histology, in a new location, complete with efferent and afferent innervations to and from the spinal cord.     

Ethanol influences on the chick embryo spinal cord motor system. II. Effects of neuromuscular blockade and period of exposure

The study described below was performed as a continuation of a previous study in which we found reduced motoneuron number in lumbar spinal cord of the chick embryo following chronic ethanol administration from embryonic day 4 (E4) to E11. We sought to determine whether this reduction was due to primary ethanol toxicity or to enhancement of naturally occurring cell death (NOCD) and to determine whether administration of ethanol at a later period of development could also reduce motoneuron number. Earlier studies have shown that curare suspends NOCD in the chick embryo. By administering both ethanol and curare to these embryos from E4 to E11 and examining the lumbar spinal cord on E12, we determined that ethanol was directly toxic to motoneurons and reduced motoneuron number in the absence of NOCD. By administering ethanol from E10 to E15 and examining the lumbar spinal cord on E16, we determined that ethanol can reduce motoneuron number without altering spinal cord length during more than one stage of chick embryo development, and that ethanol toxicity is not dependent on NOCD. In addition, we demonstrated that ethanol does not affect the neurotrophic content of chick muscle when it is administered from E10 to E15. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 684–694, 1997     

Primary afferent depolarization induced by activation of intersegmental connections of the substantia gelatinosa in the cat spinal cord

Depolarization of primary afferent terminals induced by selective activation of intersegmental connections of the substantia gelatinosa was investigated in cats anesthetized with pentobarbital. Depolarization was found to develop most rapidly in fibers of high-threshold muscular and cutaneous sensory nerves, but it was present only to a very slight degree in fibers of group Ia muscular afferents. It is suggested that the spread of activity inducing depolarization takes place in the substantia gelatinosa along a chain of excessively excited neurons. The possible role of primary afferent depolarization as a factor stabilizing coordinated activity of spinal neurons is discussed.     

Activity and synapse elimination at the neuromuscular junction

The neuromuscular junction undergoes a loss of synaptic connections during early development. This loss converts the innervation of each muscle fiber from polyneuronal to single. During this change the number of motor neurons remains constant but the number of muscle fibers innervated by each motor neuron is reduced. Evidence indicates that a local competition among the inputs on each muscle fiber determines which inputs are eliminated. The role of synapse elimination in the development of neuromuscular circuits, other than ensuring a single innervation of each fiber, is unclear. Most evidence suggests that the elimination plays little or no role in correcting for errant connections. Rather, it seems that connections are initially highly specific, in terms of both which motor neurons connect to which muscles and which neurons connect to which particular fibers within these muscles. A number of attempts have been made to determine the importance of neuromuscular activity during early development for this rearrangement of synaptic connections. Experiments reducing neuromuscular activity by muscle tenotomy, deafferentation and spinal cord section, block of nerve impulse conduction with tetrodotoxin, and the use of postsynaptic and presynaptic blocking agents have all shown that normal activity is required for normal synapse elimination. Most experiments in which complete muscle paralysis has been achieved show that activity may be essential for the occurrence of synapse elimination. Furthermore, experiments in which neuromuscular activity has been augmented by external stimulation show that synapse elimination is accelerated. A plausible hypothesis to explain the activity dependence of neuromuscular synapse elimination is that a neuromuscular trophic agent is produced by the muscle fibers and that this production is controlled by muscle-fiber activity. The terminals on each fiber compete for the substance produced by that fiber. Inactive fibers produce large quantities of this substance; on the other hand, muscle activity suppresses the level of synthesis of this agent to the point where only a single synaptic terminal can be maintained. Inactive muscle fibers would be expected to be able to maintain more nerve terminals. The attractiveness of this scheme is that it provides a simple feedback mechanism to ensure that each fiber retains a single effective input.     

Effect of laser radiation on the posttraumatic recovery of the interneuronal connections in the sympathetic trunk

Low energetic laser radiation produces stimulating influence on posttraumatic regeneration of interneuronal connections of the sympathetic trunk. Local effect of the low energetic laser with exposition of 25 sec., power of the incident ray 16 mVt/cm2 increases the cause of reparative regeneration of sympathetic preganglionic fibers. Restoration of the cranial cervical ganglion (CCG) connections with the spinal cord takes place 5-6 days earlier comparing with the control. Localization and structural organization of the spinal initial neurons is stated, their processes reach the CCG.     

Metamorphosis alters the response to spinal cord transection in Xenopus laevis frogs

A series of studies has examined the response of the spinal cord to lesions made at various stages prior to and after metamorphic climax in the clawed frog Xenopus laevis. Complete transections made between Nieuwkoop and Faber (1956) stages 50 and 62 were followed by gradual recovery of righting and coordinated swimming as animals metamorphosed into juveniles (stage 66). Examination of descending axonal projections using horseradish peroxidase (HRP) showed fibers crossing the lesion site and distributing to the caudal lumbar spinal cord. These fibers could be traced from more rostral spinal segments as well as from brainstem injections of HRP. No evidence for rostrally projecting fibers crossing the lesion was obtained. Juvenile frogs of varying ages failed to demonstrate recovery of coordinated swimming or reconstitution of spinal descending pathways. In an additional series of animals, spinal transections were made within 1 or 2 days of tail resorption to assess whether regenerative capacities extended at all into post-metamorphic stages. No evidence for regeneration was found. Studies of metamorphosing frogs after spinal transections showed that fibers crossed the lesion within 5-12 days of transection, well prior to the end of metamorphic climax; however, in some cases in which metamorphosis seemed arrested, little regeneration was observed. Immunocytochemical studies showed that fibers containing serotonin (5-HT) were included in the population of axons that rapidly crossed the lesion after transection at metamorphic stages. These results are compared to those for lesions of the dorsal columns and other systems in developing and juvenile Xenopus. It is suggested that both metamorphosis-related hormonal changes, and axon substrate pathways, may affect the regenerative response in the Xenopus central nervous system (CNS).     

Functional reinnervation of skeletal muscle in the adult rat by means of a peripheral nerve graft introduced into the spinal cord by dorsal approach

In the adult Mammal, different types of neurons, whose processes have been damaged in the CNS, may regrow lengthy axons along autologous PNS grafts. In the present study, PNS bridges were used to join the spinal cord (C5 level) to a nearby skeletal muscle (m. longissimus atlantis) which was denervated prior to direct graft insertion into an aneural region. From 3 to 5 months later, the following results were obtained: in situ and in vitro electrical stimulation of the graft determined partial or full contraction of the reconnected muscle. Intracellular recordings showed miniature endplate potentials (mepps). Endplate potentials (epps), evoked by stimulating the nerve graft, were recorded at the same points after partial blockade of the transmission with low doses of curare. They were suppressed with higher concentrations. The overall appearance of cross semithin and thin sections of the grafts was typical of regenerating nerves. EM observations of reinnervated muscles revealed typical neuromuscular junctions located either around the site of grafting or in the site of original endplates. In situ HRP application to the transected PNS bridges led to extensive labeling of neuronal somata located, close to the site of grafting, in the spinal grey matter and in adjacent spinal ganglia. When HRP was injected into the recommended muscle, neuronal labeling was almost restricted to typical motoneurons of the ventral horn. These results indicate that spinal neurons, and especially motoneurons, are probably involved in the formation, through PNS grafts, of new functional cholinergic connections with denervated skeletal muscles.     

The formation of the segmental projections of the primary afferents of the wing and leg in the chick embryo spinal cord]

A comparative HRP study of formation of connections between primary sensory nerve fibers and motoneurones in brachial and lumbosacral cord segments has been made on chick embryos between the 6.5th and 10th days of incubation. HRP was applied to the cut ends of the appropriate nerves via suction pipettes on isolated superfused spinal cord preparation. The first contacts between primary sensory collaterals and motoneuronal dendrites were found to appear both in lumbosacral and branchial cord segments at the same stage, i.e. at the 7.5-8th days of development. This observation does not confirm the widely accepted belief on rostrocaudal sequence of development of the spinal cord, indicating that exceptions from this developmental gradient are quite possible.     

Localization of substance P-like immunoreactive fibers in the thoracic spinal cord of guinea pig

Summary A dorsal-horn fiber system is revealed in the thoracic spinal cord of guinea pig by means of substance P immunocytochemistry. This system has repeated craniocaudal and/or caudo-cranial extensions and possesses five main components: (1) a superficial network, situated beneath the dorsolateral surface of the spinal cord. This network is connected with the dorsal root fibers and the accumulations of substance P-like immunoreactive (SP-LI) fibers in the Lissauer's tract; (2) an accumulation of SP-LI fibers in the Lissauer's tract at the border of the dorsal horn; (3) two collateral SP-LI fascicles (one lateral and one medial) emerging from the SP-LI fiber accumulation in the Lissauer's tract; (4) a transversal fascicle running through laminae III–V, and (5) an SP-LI network in the region of the lateral spinal cord nucleus. These components of the dorsal-horn fiber system show widespread connections with ipsi-and contralateral spinal cord areas, connecting them in cranio-caudal and/or caudo-cranial directions. The SP-LI dorsal-horn system has close relationship with groups of preganglionic sympathetic cells in the intermediate zone of the spinal cord, respective with the vegetative network of this zone. It is suggested that some fibers of the dorsal-horn system that originate from dorsal-root ganglia may represent primary sensory or visceral afferents. It is likely that the dorsal-horn fiber system and the vegetative network of the thoracic spinal cord may represent the morphological basis for the integration of (1) the central and peripheral vegetative nervous systems, and (2) the somatic and vegetative nervous system.     

Corticofugal influences on reticulospinal neurons of the gigantocellular nucleus in cats

It was shown by intracellular recording that stimulation of the motor cortex evokes E PS Ps and I PS Ps in reticulospinal neurons of the gigantocellular nucleus of the cat medulla. The E PS Ps appeared in 94.3% and the I PS Ps in 5.7% of neurons tested. Analysis of the presynaptic pathway showed that 77.4% of E PS Ps studied arose through monosynaptic, and 22.6% through polysynaptic corticoreticular connections. By their latent period, duration, and rise time up to a maximum the monosynaptic E PS Ps were divided into two groups: "fast" and "slow." It is postulated that "fast" E PS Ps are generated in reticulospinal neurons which are activated by fast-conducting fibers and "slow" E PS Ps by slowly conducting corticobulbar fibers. I PS Ps were recorded from reticulospinal neurons that also were inhibited by stimulation of the ventral columns of the spinal cord. The hypothesis is put forward that cortical motor signals in cats can be transmitted to the spinal cord via monosynaptic and polysynaptic connections of "fast" and "slow" pyramidal neurons with reticulospinal neurons.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 3, pp. 250–257, May–June, 1976.     

Use of fluorescent carbocyanine dyes in the study of the organization of the pathways in autopsy material of the human brain

Our report provides evidence that fluorescent carbocyanine dyes (diI and diO) can be used in experimental anatomical studies of the fixed autopsy human brain. The dyes transported in both anterograde and retrograde directions, providing labeling of axons with collaterals and neurons including dendrites. To study the retrograde labeling of pyramidal neurons and anterogradely labeling of afferent fibers in human motor cortex, we applied diI and diO to the white matter, I and III layers of cortex. During 2 months there was no evidence of passive diffusion from labeled fibers and neurons to other neurons or glia. This method will be useful for identifying alterations of neuronal connections associated with neurological and psychiatric disorders.     

Cholinergic innervation to the upper esophageal sphincter muscle in the eel,with special reference to drinking behavior

To elucidate innervation in the upper esophageal sphincter (UES) muscle of the eel, a key muscle in swallowing, repetitive electrical field stimulation (EFS; 30 mA, 40 V, 300 micros, 10 Hz, 10 trains) was employed. Anatomically, the eel UES muscle consists of striated fibers. The EFS-induced contraction of the UES was completely blocked by tetrodotoxin and curare, and abolished in Ca2+ -free Ringer solution. These results suggest that the EFS stimulates nerve fibers specifically and releases acetylcholine as a neurotransmitter. In fact, acetylcholine and carbachol constricted the UES in a concentration-dependent manner. Even after blocking neuronal firing with tetrodotoxin, acetylcholine constricted the UES muscle, suggesting the existence of acetylcholine receptors on the UES muscle cells. Both EFS- and carbachol-evoked contractions of the UES were blocked by curare at a lower concentration than by atropine or hexamethonium, suggesting that the acetylcholine receptor is nicotinic. Even in Ca2+ -free Ringer solution, a direct current stimulus (2 s duration) constricted the UES muscle to an extent similar to that in the presence of Ca2+, indicating that the muscle contraction itself does not need extracellular Ca2+, i.e., the muscle can be constricted by a release of Ca2+ from the sarcoplasmic reticulum.     

Muscle blood flow and fiber activity in partially curarized rats during exercise

We previously reported that low doses of d-tubocurarine attenuated glycogen loss in red muscles of rats during treadmill walking but that the initial hyperemia in the muscles was normal. The present studies were performed to 1) determine with electromyography (EMG) whether red muscle fiber activity is reduced in walking, curarized rats and 2) study muscle blood flow and glycogen loss during running with different doses of curare (dose response). At 0.5 min of treadmill walking (15 m/min), integrated EMG in vastus intermedius (VI) muscle was reduced by an average of 18% in curarized (60 micrograms/kg) rats, although blood flow (measured with microspheres) was the same as in saline control rats. Comparison of blood flows and glycogen loss in quadriceps muscles at 1 min of treadmill running (30 m/min) with different curare doses (20-60 micrograms/kg) demonstrated that red muscle glycogen loss was inversely related to curare dose but that blood flows in the same muscles were unaffected by curare. These findings provide support for our previous conclusion that at the initiation of low to moderate treadmill exercise, red muscle blood flow is not proportional to the activity or metabolism of the muscle fibers.     

Distribution of terminals of primary afferent fibers connected polysynaptically with motoneurons in the frog spinal cord

Parallel intracellular recordings of potentials in primary afferent fibers (in the region of their entry into the spinal cord) and motoneurons were made in experiments on an isolated perfused preparation of frog spinal cord preserving its connections with hind limb nerves. It was shown by injection of horseradish peroxidase through a microelectrode inserted into the fiber that fast-conducting cutaneous, tendon, and muscular afferents connected polysynaptically with motoneurons reach only the upper or middle third of the dorsal horn. Terminal branches of these fibers are characterized by numerous short terminal twigs given off at short distances apart from larger collaterals. Terminal boutons and en passant contacts, stained with horseradish peroxidase, were found on bodies of interneurons. In some cases, trans-synaptic staining of interneurons was found to take place. It is suggested that peroxidase-labeled interneurons form axo-axonal synapses with primary afferents.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 615–621, November–December, 1982.     

Efferent projections of mesencephalic locomotor neurons in the cat

Efferent neuronal projections of the mesencephalic locomotor region were investigated in cats using a horseradish peroxidase retrograde axonal transport technique. It was found that neurons located within the locomotor area form ascending and descending projections to many structures of the spinal cord and the brain but that short-axon connections running to the reticular formation of the midbrain and the medulla predominate. Small numbers of long-axon fibers may merge into the locomotor strips of the medulla and the spinal cord. The locomotor regions of the two halves of the midbrain are interlinked.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 117–125, January–February, 1986.