Degeneration and regeneration in the superior cervical sympathetic ganglion afterLatrodectus venom

Summary The effects of the venom of the spider Latrodectus mactans hasselti on the superior cervical ganglion were studied in the guinea pig. Under anaesthesia the ganglion was bathed in venom solution for 15 min. Shortly afterwards animals salivated profusely and later developed unilateral ptosis and enophthalmos. Postoperative survival times ranged from 15 min to 10 weeks. Electron microscopy showed acute swelling of preganglionic cholinergic nerve terminals, followed by degeneration with separation of synapses. Other ganglionic elements appeared to be undamaged, although after detachment of synapses the dendritic postsynaptic specializations were reduced in number. Recovery was very rapid; axon growth cones were identifiable at 18 h and synapse reformation was well established by 2 weeks. With longer survival times there was progressive restoration of normal morphology such that by 8 weeks regeneration appeared complete. These experiments indicate that the preganglionic cholinergic nerve terminals are selectively affected by Latrodectus venom and have a considerable capacity for appropriate regeneration.     

The structural basis for electrotonic coupling in the avian ciliary ganglion. A study with thin sectioning and freeze-fracturing.

Each "ciliary" neurone in the ciliary ganglion of adult birds receives its innervation from a single myelinated fibre of the oculomotor nerve by means of a dual mode of synaptic action, electrical and chemical. The preganglionic fibre branches repeatedly around the postganglionic axon but the extra-cellular compartment is large. The preterminal fibres, most of which are unmyelinated, end with large boutons on the axon hillock, a few on short dendrites and on the portion of the perikaryon of the ciliary neurone from which the axon emerges. This synaptic apparatus is enveloped by a glial sheath, mainly consisting of satellite cell bodies and loose myelin lamellae. The nonsynaptic portion of the ciliary perikaryon is covered by a sheath consisting mainly of compact myelin. The ciliary neurone has an initial axon segment like that of C.N.S. neurones. The area of each neurone apposed to boutons measures about 16,000 mum2. Approximately 9 percent is specialized for chemical transmission and 0.17 percent for electrical transmission. Each neurone has about 280,000 gap junctional particles. Assuming that each particle represents one channel, the electrical resistance provided by these junctions is estimated to be of the order of 100 k omega. The electrical coupling between the preganglionic fibre and the ciliary neurone may therefore be of resistive nature.     

Convergence on the same neurons in the feline ventrobasal thalamus of terminals from the dorsal column and the lateral cervical nuclei: an ultrastructural study combining orthograde degeneration and anterograde axonal transport of lectin conjugated horseradish peroxidase

After electrocoagulation of the dorsal column nuclei (DCN) and injections of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) into the lateral cervical nucleus (LCN), two kinds of changes in fibers and boutons were observed in the dorsolateral part of the thalamic ventroposterolateral nucleus (VPL). The axons and boutons from neurons in the DCN demonstrated dark degeneration, while the fibers and synaptic terminals from the LCN-neurons contained the characteristic needle-shaped peroxidase reaction products following incubation with tetramethylbenzidine (TMB). Occasionally dark degenerating boutons and boutons with TMB-positive reaction products showed synaptic contact with the same dendritic profiles. After consideration of the possibility of an axoaxonal transfer of the tracer it is concluded that the findings demonstrate convergence of synaptic input both from the DCN and from the LCN on the same postsynaptic VPL neurons. The present results are in accordance with earlier light microscopic and neurophysiological findings. It is also concluded that the ultrastructural technique employed in this study is well suited to reveal convergence of different afferent systems on the same postsynaptic neurons.     

Intrinsic determinants of synaptic phenotype: an experimental study of abducens internuclear neurons connecting with anomalous targets

The present experiments investigate the role of postsynaptic neurons in the morphological differentiation of presynaptic terminals that are formed de novo in the adult CNS. Abducens internuclear neurons in the adult cat were chosen as the experimental model. These neurons project onto the contralateral medial rectus motoneurons of the oculomotor nucleus. Abducens internuclear axon terminals were identified by their anterograde labeling with biocytin and analyzed at the electron microscopic level. To promote the formation of new synapses, two different experimental approaches were used. First, after the selective ablation of medial rectus motoneurons with ricin, abducens internuclear neurons reinnervated the neighboring oculomotor internuclear neurons. Second, after axotomy followed by embryonic cerebellar grafting, abducens internuclear axons invaded the implanted tissue and established synaptic connections in both the molecular and granule cell layer. Boutons contacting the oculomotor internuclear neurons developed ultrastructural characteristics that resembled the control synapses on medial rectus motoneurons. In the grafted cerebellar tissue, abducens internuclear axons and terminals did not resemble climbing or mossy fibers but showed similarities with control boutons. However, labeled boutons analyzed in the granule cell layer established a higher number of synaptic contacts than controls. This could reflect a trend towards the mossy fiber phenotype, although labeled boutons significantly differed in every measured parameter with the mossy fiber rosettes found in the graft.We conclude that at least for the abducens internuclear neurons, the ultrastructural differentiation of axon terminals reinnervating novel targets in the adult brain seems to be mainly under intrinsic control, with little influence by postsynaptic cells.     

Direct synaptic projections to the myenteric ganglion of the rat subdiaphragmatic esophagus from the dorsal motor nucleus of the vagus

We have examined the ultrastructure of the myenteric ganglion of the subdiaphragmatic esophagus and determined whether the ganglion neurons receive direct projections from the dorsal motor nucleus of the vagus (DMV) using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) as an anterograde tracer. The neurons (22.2 microm x 13.3 microm) of myenteric ganglion in the esophagus contained dark cytoplasm having many free ribosomes, mitochondria, and an oval nucleus, and received only a few axon terminals contacting somata. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Approximately 85% of the axon terminals contacting dendrites and about 50% of the axon terminals contacting somata contained pleomorphic vesicles, while the rest contained round synaptic vesicles. When WGA-HRP was injected into the DMV, anterogradely labeled fibers and terminals were found in the myenteric ganglia. The WGA-HRP labeled terminals were large (1.97 microm) and contained round clear vesicles and small granular vesicles. These labeled terminals contacted exclusively the small dendrites, but not the somata. These results suggest that the DMV neurons project directly to the myenteric ganglion neurons and regulate the esophageal muscles via the ganglion neurons.     

Ultrastructure of motor end-plates during pharmacologically-induced degeneration and subsequent regeneration of skeletal muscle

Summary A local anaesthetic, methyl-bupivacaine was injected into the planta of adult mice, and the ultra-structure of motor end-plates was studied during the degenerative and regenerative cycle induced in lumbrical muscles. Muscle degeneration took place during the first day after drug administration. The postsynaptic part of the neuromuscular junction completely degenerated as did the whole injured muscle fibre. Nerve terminals, however, remained unaffected. By the second day after muscle injury, axon terminals were enclosed within Schwann cell cytoplasm and thus became separated from the residual sarcolemmal tube. One to three days later, when myotubes were formed by fusion of the surviving myoblasts, the layer of Schwann cell cytoplasm on nerve terminals was discontinuous. Subsequently nerve terminals approached the regenerating muscle cell and the subneural apparatus began to differentiate. Slight depressions and furrows appeared on the myotube surface below the nerve ending and the myotube membrane, covered with basement membrane, became undercoated by dense material in this region. Where the distance between nerve ending and myotube was reduced to that found in the normal neuromuscular junction, i.e. to about 500 Å, junctional folds were formed. Fourteen days after drug administration, newly formed end-plates were indistinguishable from those in normal control lumbrical muscles.     

Ultrastructural changes in the chick thymus following unilateral vagotomy.

The ultrastructure of the thymus in the chick (Gallus domesticus) was studied after unilateral vagotomy at survival times of 3, 7 and 10 days. Ultrastructural changes in the ipsilateral thymus were observed in axon boutons as well as in myoid and cystic cells in the medulla, especially those situated near the corticomedullary junction. Structural changes in axon boutons ranged from granular degeneration of the axonal cytoskeleton to vacuolation of the axoplasm. Myelin figures of different sizes and configurations and clumping of small agranular vesicles were commonly observed in the axon terminals. Degeneration of myoid cells appeared to peak at 7 days post-vagotomy. Changes ranged from oedematous appearance and intense vacuolation of the peripheral cytoplasm to disorganisation and clumping of myofibrils. In some myoid cells the sarcomeres showed granular degeneration at the I-bands and in others, the myofibrils were completely degenerated such that amorphous material and partially degenerated organelles filled the entire cell. The majority of cystic cells at 3 days post-vagotomy showed a uniform increase in electron density. Numerous electron dense bodies, some displaying concentric lamellation, were observed throughout the expanse of the cytoplasm. At 7 days post-vagotomy, the cytoplasm of some cells gave a "moth-eaten" appearance. Dying cystic cells were encountered at 10 days after vagotomy. Degeneration in the myoid and cystic cells suggests that these cellular components may be the putative targets of the vagal fibres in the chick thymus. The changes in these cells reflect a disturbance in the cell metabolism presumably brought about by the removal of vagal influence.     

Vestibulospinal projections in the toad. An experimental electron microscopic study

An experimental electron microscopical study has been made on the mode of termination of the vestibulospinal projections in the toad. Degenerating fibers and boutons were observed in the ventromedial region of the spinal cord in which five types of terminals were identified in normal preparations: S-type, F-type, C-type, NfS-type and NfF-type. The latter two types represent the largest terminals in the spinal gray. In the medial region of the ventral horn they seem to belong to supraspinal fibers.Following electrolytic lesions of the dorsal and ventral nuclei nervi octavi, some of the NfF-type of boutons undergo filamentous degeneration which progresses into the dark type at longer survival periods (7–20 days). In two animals, a few NfS-type of boutons also display filamentous hypertrophy.Problems connected with the filamentous type of degeneration in amphibians and with the identification of bouton types belonging to the vestibulospinal pathway are discussed.Degenerating fibers and boutons are seen at all levels of the spinal cord, ipsilateral and also, in smaller number, contralateral to the lesions. The vestibulospinal fibers terminate predominantly on large and small dendrites, and only occasionally on the somata of large neurons on the ipsilateral side.     

Neuron morphology and synaptic architecture in the medial superior olivary nucleus

Summary Dendritic arborization pattern, spatial and synaptic relations of various neuron types and the terminal distribution of afferent axons of various origin were studied in the medial superior olivary nucleus of the cat using Golgi, degeneration, electron microscope and horseradish peroxidase techniques. Three types of neurons clearly different in morphological features, distribution, neighbourhood relations, input and output characteristics were distinguished: (1) fusiform cells having specific dendritic orientations and arborization patterns and synaptic relations to various types of terminal axon arborizations (2) multipolar neurons with wavy dendrites bearing spine-like appendages, receiving relatively few synaptic contacts and having a locally arborizing axon, and (3) elongated marginal cells, largely restricted to the fibrous capsule of the nucleus. The fusiform and marginal neurons were identified by retrograde peroxidase labeling as the olivo-collicular projection cells.Ultrastructural analysis of normal and experimental material revealed the presence of four distinct kinds of axon terminals differing in size, synaptic vesicles type, relation to postsynaptic targets and in origin: (i) large terminals with multiple extended asymmetric synaptic membrane specializations and containing round, clear vesicles arise from the spherical cells of the ipsilateral anteroventral cochlear nucleus, (ii) most of the small axon terminal profiles — engaged in asymmetric synaptic contacts — originated from the trapezoid nucleus, (iii) terminal boutons containing pleomorphic vesicles belong to fibers descending from the ipsilateral multipolar neurons in the central nucleus of the inferior colliculus and from the nuclei of the lateral lemniscus while (iv) boutons containing exclusively ovoid vesicles and remaining intact after complete deafferentation of the nucleus were considered to be of local origin.     

Morphological aspects of the elimination of polyneuronal innervation of skeletal muscle fibres in newborn rats

Summary The ultrastructure of neuromuscular junctions of rat soleus muscles 1–40 days postnatally was examined for possible morphological correlates of the transient polyneuronal innervation which is present in newborn rats.Several vesicle-laden profiles of terminal axons are seen to contact each muscle fibre up to 8 days postnatally. Axon terminals often lie close together, without Schwann cell intervention. Between days 8 and 16 the number of profiles of terminals on each muscle fibre is reduced, and both Schwann cells and ridge-like extensions of muscle fibre cytoplasm intervene between and separate axon terminals.No signs of degenerating intramuscular axons or axon terminals could be found. It is suggested that the redundant terminals are eliminated by retraction into the parent axons. This process is apparently accomplished without any morphological signs of degeneration.     

Ultrastructural identification of degenerating boutons of monosynaptic pathways to the lumbosacral segments in the cat after spinal hemisection

Summary Electrophysiological results suggest that the supra-spinal monosynaptic pathways to the lumbosacral motoneurones in cats are located in the vestibulospinal and reticulospinal tracts. In the present study these pathways were interrupted in the upper thoracic region by hemisection of the spinal cord. The boutons undergoing degeneration were then studied both qualitatively and quantitatively by means of serial and single sections of selected areas of the ventral horn in spinal cord segments L 6, L 7 and S 1. The degenerating boutons were distributed over the cell soma and larger dendrites (but not the most proximal portions) of the motoneurones. Two days postoperatively boutons showing signs of degeneration were readily identified in the latero-ventral motoneurone nucleus and Rexed's lamina VIII. These boutons which were swollen had a greatly increased number of neurofilaments and a decreased number of synaptic vesicles. Five days postoperatively these boutons which were now shrunken, contained swollen mitochondria and a dense, granular material. Between 5 and 8 days post-operatively the boutons were invaded by glial cell processes and digested. A small percentage of the F- and S-type boutons (approximately 0.5 and 3.0% respectively) were identified as forming the terminals of the experimentally interrupted pathways. This study did not exclude the possibility that a proportion of these boutons were the terminals of the propriospinal, monosynaptic fibers derived from the cervical region of the cord.On sabbatical leave from the Department of Zoology, University of Melbourne     

Reciprocal synapses between cholinergic axons and small granule-containing cells in the rat cardiac ganglion

Electron microscopy of the rat cardiac ganglion shows occurence of small granule-containing cells that form reciprocal synaptic junctions with cholinergic terminals. At the synaptic junctions which are from axon to granule-containing cell, the intraaxonal vesicles are clustered against the junctional axolemma, but dense-cored vesicles in the postynaptic cell do not cluster towards the membrane densities in these synapses. By contrast, the synaptic zone polarized in the opposite direction shows an absence of axonal vesicles in close proximity to the postsynaptic axolemma, but there is a marked aggregation of dense-cored vesicles towards the presynaptic specializations of granule-containing cells. The synaptic zones are multifocal rather than bifocal, and the minimal distance separating each synaptic zone is about 0.3 μ. These findings may indicate that cholinergic excitation of some or all granule-containing cells causes a reciprocal inhibition of one or more cholinergic terminals.     

Ultrastructural alterations induced in quail ciliary neurons by postganglionic nerve crush and by Ricinus toxin administration, separately and in combination

The response to postganglionic nerve crush and Ricinus toxin administration by the ciliary neurons of the quail ciliary ganglion was investigated at the ultrastructural level. The toxin was either applied at the crush site on the postganglionic nerves or injected into the anterior eye chamber without any other operative intervention. Crush of postganglionic nerves without toxin administration and saline injection into the anterior eye chamber served as controls for the two toxin administration procedures. Postganglionic nerve crush caused a distinct chromatolytic reaction, accompanied by massive detachment of the preganglionic axon terminals from the ciliary neurons and loss of most of the synapses, both chemical and electrical. This process does not induce cell death and is reversible. Saline injection in the anterior eye chamber caused a moderate retrograde reaction in some of the ciliary neurons, presumably as a consequence of paracentesis. The changes consisted mainly of an increase of perikaryal neurofilaments with, at most, a minor detachment of the preganglionic boutons from a small portion of the cell body at the nuclear pole. Ricinus toxin administration induced neuronal degeneration following a pattern common to both delivery modes. The degenerative process consisted of disruption and detachment of polyribosomes from the rough endoplasmic reticulum, an increase of smooth cisterns and tubules, a dramatic increase of neurofilament bundles, compartmentalization of the cytoplasmic organelles and, finally, karyorrhexis and cell lysis. The final stages of Ricinus toxin degeneration involve a progressive accumulation of extracellular flocculo-filamentous material and cell lysis. After administration of Ricinus toxin to the crush site, ricin-affected neurons showed withdrawal of the preganglionic boutons from a portion of the ciliary neuron, especially at the nuclear pole. After Ricinus toxin injection into the anterior eye chamber, however, the bouton shell surrounding the affected ciliary neurons remained intact in the early stages of degeneration. Detachment of the preganglionic terminals and disruption of the cell junctions, therefore, is the consequence of nerve crush and not of the toxin itself.

This study demonstrates that quail ciliary neurons are a suitable model for experimental neuropathology and neurotoxicology.     

Degeneration and regeneration of frog ganglionic synapses.

In the sympathetic neurons of the frog, the majority of both the postsynaptic ‘membrane thickenings’ and the subsynaptic dense layers disappear in the first few days after section of preganglionic fibres. However, a limited number of both these specializations may survive for a long time (at least 2 months). In the same experimental conditions, the regeneration of ganglionic synapses was investigated after variable survival periods of up to 2 months. A maximum of about 10% of the normal number of synapses was found, perhaps due to the difficulty of penetrating the glial sheath. It is suggested that a large proportion of the postsynaptic specializations may be newly formed by the action of the presynaptic endings.     

Membrane ultrastructure of the giant synapse of the squidLoligo pealei

The ultrastructure of the ‘giant synapse’ of the stellate ganglion of the squid was studied with freeze-fracture and thin-sectioning techniques. A sheath of glial cells separates the pre- and post-synaptic axons. At intervals, round-topped processes of the postsynaptic axon pierce the sheath to contact the presynaptic axon. This area of synaptic contact is marked by a widened intercellular cleft containing electron-dense material and by a cluster of synaptic vesicles within the presynaptic cytoplasm. The number of synaptic vesicles in such clusters was greatly reduced by electrical stimulation of the synapse during fixation. Freeze-fracture reveals a roughly circular patch (0.3 μm diameter) of 10 nm particles on the cytoplasmic leaflet of the presynaptic membrane. A similar patch of particles lies on the external leaflet of the apposed postsynaptic membrane.The squid giant synapse thus consists of multiple small pre- and postsynaptic active zones where neurotransmitter is released from the presynaptic terminal and sensed by postsynaptic receptors. Comparison of the structure of these postsynaptic active zones with those at synapses where the transmitter or transmitter action is known suggests that the excitatory transmitter at this synapse is an amino acid.Presumptive gap junctions, marked by particles in the cytoplasmic leaflet, are found between small-diameter axons in the stellate ganglion but not at the giant synapse. Glial-cell membranes contain aggregates of particles and pits suggestive of gap junctions. The aggregates of pits are embedded within linear arrays of particles which somewhat resemble tight junctions.     

Ultrastructural changes in the chick thymus following unilateral vagotomy

The ultrastructure of the thymus in the chick (Gallus domesticus) was studied after unilateral vagotomy at survival times of 3, 7 and 10 days. Ultrastructural changes in the ipsilateral thymus were observed in axon boutons as well as in myoid and cystic cells in the medulla, especially those situated near the corticomedullary junction. Structural changes in axon boutons ranged from granular degeneration of the axonal cytoskeleton to vacuolation of the axoplasm. Myelin figures of different sizes and configurations and clumping of small agranular vesicles were commonly observed in the axon terminals. Degeneration of myoid cells appeared to peak at 7 days post‐vagotomy. Changes ranged from oedematous appearance and intense vacuolation of the peripheral cytoplasm to disorganisation and clumping of myofibrils. In some myoid cells the sarcomeres showed granular degeneration at the I‐bands and in others, the myofibrils were completely degenerated such that amorphous material and partially degenerated organelles filled the entire cell. The majority of cystic cells at 3 days post‐vagotomy showed a uniform increase in electron density. Numerous electron dense bodies, some displaying concentric lamellation, were observed throughout the expanse of the cytoplasm. At 7 days post‐vagotomy, the cytoplasm of some cells gave a “moth‐eaten” appearance. Dying cystic cells were encountered at 10 days after vagotomy. Degeneration in the myoid and cystic cells suggests that these cellular components may be the putative targets of the vagal fibres in the chick thymus. The changes in these cells reflect a disturbance in the cell metabolism presumably brought about by the removal of vagal influence. Anat Rec 255:261–270, 1999. © 1999 Wiley‐Liss, Inc.     

Neonatal nerve growth factor treatment alters the preganglionic innervation pattern of rat superior cervical ganglion.

We treated rat pups with nerve growth factor (10 micrograms/animal/day s.c.) over postnatal days 1-7. Subsequent adult neuron numbers and tyrosine hydroxylase content in superior cervical ganglion were normal, but preganglionic inputs, as gauged from ganglionic choline acetyltransferase, were reduced. In parallel, intraganglionic axon terminals containing calcitonin gene-related peptide, but not those containing substance P, were increased in number. We postulate that neonatal nerve growth factor stimulates sprouting of ingrowing axons that have entered the ganglion soon after birth and that this represses subsequent establishment of cholinergic preganglionic synapses.     

Ultrastructural development of the dorsal lateral geniculate nucleus of genetically microphthalmic mice

Summary The ultrastructure of the dorsal lateral geniculate nucleus (dLGN) of microphthalmic mice is described in affected white homozygotes (mi/mi) and their apparently normal grey littermates. In the dLGN of mi/mi animals populations of apparently normal axon terminals were observed, including some with flattened synaptic vesicles and other small terminals with round vesicles and dark mitochondria (RSD), possibly of cortico-thalamic origin, just as in normal mice. However, no typical large retinal endings with round vesicles and pale mitochondria (RLP) are visible. Instead they appear to be replaced by other large boutons with round vesicles and dark mitochondria (RLD). Eye enucleation does not cause degeneration of these RLD terminals. In apparently normal grey littermates RLP terminals are present and they degenerate when an eye is enucleated. But RLD endings are also found in these animals, and never degenerate after enucleation. The origin of the RLD terminals is unclear but seems not to be cortical. These findings are compared with those of Cullen and Kaiserman-Abramof (1976) in a different strain (ZRDCT-An) of anophthalmic mouse in which they found large replacement terminals similar to our RLD boutons.     

Acute ultrastructural and behavioral effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice

Acute actions of MPTP on behavior and on neostriatal ultrastructure were examined in young C57 Black mice. Autonomic, motor, and toxic effects of MPTP exhibited dependence on dose (20-40 mg/kg) and time during the first 4 h after subcutaneous injection. The ultrastructure of the neostriatum was altered very quickly (2-24 h) after single injections of MPTP. Darkened glial processes were found within 2-8 h, followed by dark degeneration of synaptic boutons, especially those making small symmetric synapses. More rarely, swollen axons and postsynaptic degeneration were also observed.