Regeneration of neuromuscular connections in crayfish allotransplanted neurons

Transplantation of whole ganglia was used to study the regeneration of four of the neurons that innervate the superficial flexor muscles of the crayfish Procambarus clarkii. The isolated ganglia containing the somas of these neurons were successfully transplanted from one crayfish to another. Reinnervation proceeded across the muscle surface and by 8 to 10 weeks connections were detected across the entire target field. At different time periods after the transplant, junction potentials (JPs) produced in phase with spontaneous neuronal spikes were recorded. The distribution of JP sizes and their decay times were examined. JPs from transplanted preparations were smaller than JPs from control or normal regeneration animals. These JPs also failed to facilitate when stimulated at 1 and 10 Hz. These are normal characteristics of immature terminals, but in the transplant preparations, once established, they remained stable for the duration of the study. Thus, synaptogenesis appears to be arrested at a stage before synaptic efficacy is established in the allotransplants. In addition, connectivity maps were plotted for each axon over the muscle surface. Some muscle fibers did not receive any contacts, and overall innervation leveled off at around 60% of the muscle fibers, remaining stable for the duration of this study. Despite the incomplete physiological innervation, however, three of the four neurons showed the same medial/lateral preferences observed in control animals, regenerating their original patterns of connectivity across the muscle surface. © 1995 John Wiley & Sons, Inc.     

Synaptic plasticity in a regenerated crayfish phasic motoneuron

Crustacean neuromuscular systems provide many advantages for the study of synaptic transmission and plasticity. The present study examines aspects of synaptic transmission in the phasic, fast closer excitor (FCE) motoneuron of regenerated crayfish claws. Excitatory postsynaptic potentials (EPSPs) fatigued rapidly and showed poor long-term facilitation (LTF) in the smallest of regenerating claws. EPSPs in larger regenerating claws fatigued less and showed pronounced facilitation. These observations were not the same as those previously made during primary development of this motoneuron (Lnenicka and Atwood, 1985a, J. Neuroscience 5:459–467). Hence, regeneration is not the recapitulation of primary development. In situ stimulation of the FCE is known to lead to long-lasting adaptation of synaptic performance. This adaptation is age dependent; it is expressed in young but not old animals. In the regenerated FCE of old animals, we observed a novel form of long-lasting adaptation to imposed activity: EPSPs showed large initial EPSPs and did not exhibit resistance to fatigue during maintained stimulation. This indicates that aged motoneurons can express adaptive changes to increased activity following axonal regeneration, but that the adaptive changes are the opposite to what is observed in nonregenerated motoneurons. © 1992 John Wiley & Sons, Inc.     

Physiological activity-dependent ultrastructural plasticity in normal adult rat neuromuscular junctions

The major finding of the present study is that the ultrastructural organization of the neuromuscular synapse can be modified by a small, 4-week-long, physiological increase in the locomotor activity of the extensor digitorum longus muscle of normal adult rats trained to walk. This study measures these plastic adaptations using several synaptic morphological parameters. The observed changes in neuromuscular junctions affect both pre- and postsynaptic membranes. In particular, the presynaptic membrane densities in the active zones and the postsynaptic adaxonal membrane densities become larger, which shows that in the normal adult mammal neuromuscular junction, there is an activity-dependent modulation of the neurotransmission-related structures in response to slight physiologic functional demands. The nature and magnitude of these changes are discussed.     

Reformation of the pattern of neuromuscular connections in the regenerated axolotl hindlimb

Retrograde neuronal tracing with horseradish peroxidase (HRP) was used to determine the position in the spinal cord of motor neurone pools innervating muscles in the regenerated axolotl hindlimb. This method allows a detailed analysis of the accuracy of reformation of neuromuscular connections. The results show that regenerated distal limb muscles are reinnervated by motor neurones in the same region of the cord as those that innervate normal control distal limb muscles but that proximal muscles are innervated by a mixture of motor neurones in a normal position and motor neurones in a region of the spinal cord that normally supplies innervation to distal limb muscles. This difference between the reinnervation of proximal and distal limb muscles suggests that axons destined for proximal muscles may not enter distal limb territory during reinnervation of the regenerated limb.     

Agrin and acetylcholine receptors are removed from abandoned synaptic sites at reinnervated frog neuromuscular junctions

Changes in the distribution of agrin and acetylcholine receptors (AChRs) were examined during reinnervation and following permanent denervation as a means of understanding mechanisms controlling the distribution of these molecules. Following nerve damage in the peripheral nervous system, regenerating nerve terminals preferentially return to previous synaptic sites leading to the restoration of synaptic activity. However, not all portions of original synaptic sites are reoccupied: Some of the synaptic sites are abandoned by both the nerve terminal and the Schwann cell. Abandoned synaptic sites contain agrin, AChRs, and acetylcholinesterase (AChE) without an overlying nerve terminal or Schwann cell providing a unique location to observe changes in the distribution of these synapse-specific molecules. The distribution of anti-agrin and AChR staining at abandoned synaptic sites was altered during the process of reinnervation, changing from a dense, wide distribution to a punctate, pale pattern, and finally becoming entirely absent. Agrin and AChRs were removed from abandoned synaptic sites in reinnervated frog neuromuscular junctions, while in contralateral muscles which were permanently denervated, anti-agrin and AChR staining remained at abandoned synaptic sites. Decreasing synaptic activity during reinnervation delayed the removal of agrin and AChRs from abandoned synaptic sites. Altogether, these results support the hypothesis that synaptic activity controls a cellular mechanism that directs the removal of agrin from synaptic basal lamina and the loss of agrin leads to the dispersal of AChRs. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 999–1018, 1997     

Systems of chemoperception in Decapod crayfish

The review presents data on some peripheral and central structures in the system of perception of chemical stimuli in crayfish and other Decapoda. The hair receptors on chelipeds, antennas, and antennules are innervated by mechano- and chemoreceptor neurons. Antennules are crayfish specialized chemoreceptor organs whose surface contains groups of exteroceptors. On claws of ambulatory feet (AF), antennas, antennules, and other mobile appendages there is a regular disposition of exteroceptor receptive fields in the form of receptor hair bushes. Behavioral experiments have shown sensitivity of crayfish to odor of individuals of their gender and sex partners as well as the presence in crayfish of pheromones providing connection of female with offspring at the initial stages of their life cycle. Individual chemosensory cells innervating hair bushes on the crayfish AF respond to amino acids, amines, nucleotides, and sugars. Minimal thresholds of reaction of the studied Decapoda chemoreceptors in response to some chemical compounds correspond to 0.1–1.0 μM. For some chemoreceptors, dose-dependent effects have been shown. Alongside with monomodal chemoreceptors, the crayfish have bimodal receptors perceiving mechanical and chemical stimuli. The efficient response of crayfish chemoreceptors can be obtained to the substance that has amino group with hydrogen bridge to carboxyl group, contains no more that 3 carbon atoms in the chain, is characterized by a certain stereoform. Among chemoreceptors there are fast and slow adapting cells. Efficiency of response of individual chemoreceptors depends on temperature of medium. In crayfish, chemoreceptors responding to ecdysterons have been revealed. Ecdysterons play a great role in intra- and inter-species communications in Crustacea. Based on the study of efferent responses of interneurons of the first and higher orders in the first thoracic crayfish ganglion to stimulation of the own receptive fields, a concept has been put forward of the structural-functional organization of afferent projections at the segmental level. Peculiarities of afferent projections from antennule chemoreceptors are considered. The data are presented on connections of these chemoreceptors with antennular, olfactory, and accessory lobules, various cell groups, interneurons of the first, second, and third orders located in various brain parts. An attention has been drawn to connections of serotonin neurons in glomeruli with endings of chemoreceptor neurons and projections of interneurons of the higher orders, which are located in the internal medulla of the crayfish eyestalks. Several principles of integration of the chemoreceptor information in central parts of the crayfish nervous system are discussed. The giant serotonin neurons revealed in crayfish glomeruli most likely participate in formation of memory to certain chemical actions. Polymodal receptor signals in the central chain of the perception system activate autonomic centers, and the changes of the animal functional state can be evaluated from the heart responses. These responses recorded by novel noninvasive methods allow detection of the initial and other phases of the stress state at changes of the medium chemical quality. Progress of the current biochemical and electrophysiological methods of study chemoreceptors allow hoping for learning of fine chemoperception mechanisms in invertebrate and vertebrate animals.     

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.     

Presynaptic to postsynaptic relationships of the neuromuscular junction are held constant across age and muscle fiber type

The neuromuscular junction (NMJ) displays considerable morphological plasticity as a result of differences in activity level, as well as aging. This is true of both presynaptic and postsynaptic components of the NMJ. Yet, despite these variations in NMJ structure, proper presynaptic to postsynaptic coupling must be maintained in order for effective cell‐to‐cell communication to occur. Here, we examined the NMJs of muscles with different activity profiles (soleus and EDL), on both slow‐ and fast‐twitch fibers in those muscles, and among young adult and aged animals. We used immunofluorescent techniques to stain nerve terminal branching, presynaptic vesicles, postsynaptic receptors, as well as fast/slow myosin heavy chain. Confocal microscopy was used to capture images of NMJs for later quantitative analysis. Data were subjected to a two‐way ANOVA (main effects for myofiber type and age), and in the event of a significant (p < 0.05) F ratio, a post hoc analysis was performed to identify pairwise differences. Results showed that the NMJs of different myofiber types routinely displayed differences in presynaptic and postsynaptic morphology (although the effect on NMJ size was reversed in the soleus and the EDL), but presynaptic to postsynaptic relationships were tightly maintained. Moreover, the ratio of presynaptic vesicles relative to nerve terminal branch length also was similar despite differences in muscles, their fiber type, and age. Thus, in the face of considerable overall structural differences of the NMJ, presynaptic to postsynaptic coupling remains constant, as does the relationship between presynaptic vesicles and the nerve terminal branches that support them. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 744–753, 2013     

The cerebral ganglia of Milnesium tardigradum Doyère (Apochela, Tardigrada): Three dimensional reconstruction and notes on their ultrastructure

Differential interference contrast micrographs from stretched animals, serially sectioned semi-thin and ultrathin sections revealed that the cerebral ganglia (supraoesophageal mass) of the eulardigrade Milnesium tardigradum lie above the buccal tube and adjacent tissue like a saddle. It has an anterior indentation which is penetrated by two muscles that arise from the cuticle of the forehead. The cerebral ganglia consist of lateral outer lobes bearing an eye on each side, and two inner lobes which extend caudally. Between the inner lobes a cone-like projection tapers into a nerve bundle. Each outer lobe is joined with the first ventral ganglion. From the outer lobe near the eye the ganglion for a posterolateral sensory field extends to the epidermis. Anterior to the supraoesophageal mass are three dorsal ganglia for the upper three peribuccal papillae. Two additional ganglia attached to the cerebral mass supply the lateral cephalic papillae. The cerebral ganglia are covered by a thin neural lamella. The pericarya which surround the neuropil have large nuclei. Near the axons in the centre of the supraoesophageal mass the cytoplasm is crowded with vesicles of different size and appearance. Some of them resemble synaptic vesicles while others resemble dense core bodies. Structurally different types of synapses and axons can be distinguished within the neuropil.     

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.     

Tracing of neurosecretory neurons in crayfish optic ganglia by cobalt iontophoresis

Summary The axonal connections between the medulla terminalis ganglionic X-organ (MTGXO) and the sinus gland are traced by iontophoretic application of cobalt dye to the neurosecretory system in the eyestalks of the crayfish, Orconectes limosus. The MTGXO consists of about 15 large perikarya, forming a distinct subgroup of neurosecretory cells in the medulla terminalis and giving rise to a prominent fibre bundle. Additional axons reaching the sinus gland from the medulla interna, the medulla externa and the optic nerve are less conspicuous.Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 87, Projekt A 3).Part of the work has been presented at the 9th Conference of European Comparative Endocrinologists in Giessen, August 1977Thanks are due to Dr. H.G. Wolff of the Universität Köln for his advice during the initial stage of this work     

Differential delay of reinnervating axons alters specificity in the rat serratus anterior muscle

Previous studies have shown remarkable rostrocaudal selectivity by regenerating motoneurons to the rat serratus anterior (SA) muscle after freezing, crushing, or sectioning the long thoracic (LT) nerve. The LT nerve contains motoneurons from both the sixth and seventh cervical spinal nerves (C6 and C7), with C6 motoneurons as the major source of innervation throughout the muscle, and with C7 motoneurons innervating a larger percentage of muscle fibers caudally than rostrally. To determine if synaptic competition can play a role in neuromuscular topography, both the LT nerve and the branch carrying C6 (rostral) motoneurons to the LT nerve were crushed in newborn rats. This approach provides a temporal advantage to regenerating C7 (caudal) motoneurons. After an initial period during which C7 motoneurons reinnervated a larger proportion of muscle fibers than normal in all SA muscle sectors, C6 motoneurons regained their original proportion of rostral muscle fibers. Caudally, however, C7 motoneurons maintained an expanded territory. With this two-site crush method, the number of C6 motoneurons that reinnervate the SA muscle was significantly decreased from normal, whereas the number of C7 motoneurons remained the same. It is concluded that when C7 motoneurons are given a temporal advantage, synaptic specificity can be altered transiently in rostral muscle sectors and permanently in caudal sectors, and this is correlated with a disproportionate loss of C6 motoneurons. Moreover, this may be an important model for studies of synaptic competition, where terminals destined to be eliminated can be identified beforehand. © 1995 John Wiley & Sons, Inc.     

Synaptic connections between sensory afferents and the common inhibitory motoneuron in crayfish

The sensory inputs to the common inhibitory motoneuron that innervates every leg muscle of the crayfish Procambarus clarkii (Girard) were analyzed by performing intracellular recordings from its neurite within the neuropil of the 5th thoracic ganglion. Two types of sensory inputs involved in locomotion were studied, those from a movement coding proprioceptor (the coxobasal chordotonal organ) and those from sensory neu rons coding contact forces exerted at the tip of the leg on the substrate (the dactyl sensory afferents). Sinusoidal movements applied to the chordotonal organ strand induced a stable biphasic response in the common inhibitory motoneuron that consisted of bursts of spikes during release and stretch of the strand, corresponding to raising and lowering of the leg, respectively. Using ramp movements imposed on the chordotonal strand, we demonstrated that only movement-coding chordotonal afferents produce excitatory post-synaptic potentials in the common inhibitory motoneuron; these connections are monosynaptic. Mechanical or electrical stimulation of the dactyl sensory afferents resulted in an increase in the tonic discharge of the common inhibitory motoneuron through polysynaptic excitatory pathways. These two types of sensory cues reinforce the central command of the common inhibitory motoneuron and contribute to enhancing its activity during leg movements, and thus facilitate the relaxation of tonic muscle fibres during locomotion.Abbreviations ADR anterior distal root - A Lev anterior levator nerve - CB coxo-basipodite joint - CBCO coxo-basal chordotonal organ - CI common inhibitory motoneuron - Dep depressor nerve - DSA dactyl sensory afferents - EPSP excitatory post-synaptic potential - IN interneuron - MN motoneuron - PDR posterior distal root - P Lev posterior levator nerve - Pro promotor nerve - Rem remotor nerve     

A model of selective synapse formation in sympathetic ganglia

In the sympathetic system, neurons from several spinal segments are mapped onto targets in the periphery in a topographically ordered way by means of selective synaptic connections in the superior cervical ganglion. Experimental evidence points to a crucial role for chemoaffinity in establishing this topographic map. Furthermore, rearrangements of synapses after surgical manipulations indicate that this chemoaffinity is not based on rigid “key-and-lock” markers. Our model is used to study how such nonrigid markers may interact with other regulatory factors, including growth-regulating signals and the growth potential of individual nerons. In the model, these latter factors are limiting, so that an increasing number of synaptic contacts decreases the likelihood of further synapse formation. These factors are combined with chemoaffinity using a linear threshold model. The model is robust to parameter changes and reproduces experimental observations with reasonable detail. Simulation results are used to discuss characteristic experimental results, such as the substantial plasticity of the connections seen after partial denervation. A surprisingly small effect of transient hyperinnervation in the model may help explain why final connectivities are similar in two real situations with high and low degrees of transient hyperinnervation (development and adult reinnervation). It is shown that spatial restrictions on post-synaptic neurons (dendrites) may contribute significantly to the segmentally broad innervation of each ganglion cell. Finally, we discuss potential effects of presynaptic neuronal death in systems with a high degree of plasticity. © 1993 John Wiley & Sons, Inc.