Ultrastructure of neuromuscular junctions in Drosophilal: Comparison of wild type and mutants with increased excitability

The ventral longitudinal muscles of the Drosphila larval body wall are innervated by at least four types of synaptic terminals that can be distinguished on morphological grounds at the light microscopical level. The innervation of these muscles has been previously shown to be regulated by neuronal activity. In this report we investigate the ultrastructural basis for synaptic bouton differences by using serial sections, and examine the structure of synaptic terminals in mutants with increased excitability. We report that individual identifiable muscle fibers are innervated by terminals containing two to three types of synaptic boutons that can be distinguished in terms of synaptic vesicle population, presynaptic and postsynaptic specialization, and general shape. We propose a model to account for the bouton types observed at the light microscopical level. We find that in the hyperexcitable mutant eag Sh, there are dramatic ultrastructural alterations at synaptic boutons. These alterations include a partial depletion of two types of synaptic vesicles and a change in appearance of a third type, changes in number and appearance of synaptic densities, and the presence of multivesicular bodies. Our results show that an increase in neuronal excitability produces profound effects in synaptic terminal structure. © 1993 John Wiley & Sons, Inc.     

Structural plasticity at crustacean neuromuscular synapses

Crustacean motor axons innervate muscle fibers via a multiplicity of synaptic terminals which release small but variable amounts of transmitter. Differences in release performance appear to be correlated with the size of synaptic contacts and presynaptic dense bars (active zones). These structural parameters proliferate via sprouting from existing synaptic terminals and relocate to ever more distal sites during development and growth of an identified axon. Moreover, alterations in number of synaptic contacts and active zones occur in adults following stimulation or decentralization, demonstrating structural plasticity of crustacean neuromuscular synapses.     

Morphometric analysis of circadian variations in the retinal photoreceptor synaptic terminals of the adult and fetal guinea pig

In order to test whether the alterations in photoreceptor synaptic terminal size and shape reported in lower vertebrates occur in a mammalian visual system, adult and fetal guinea pig retinas were exposed to an LD 12:12 lighting cycle, as well as to long-term light (LL) and long-term dark (DD) regimes. Representative random samples from all retinal quadrants, obtained at various times during these lighting regimes, were processed for electron microscopy. The synaptic terminals of all three photoreceptor cell types in this retina (alpha and paranuclear rods, and cones) were analyzed with computer-assisted morphometrics for changes in their area, perimeter, synaptic vesicle density, and the degree of plasmalemmal infolding. The data showed all three types of adult receptor terminals to have increased area and vesicle density, as well as decreased membrane infolding, during the light period, while both types of rods showed increased perimeter measurements in the dark. Results from adults maintained under extended lighting conditions (LL and DD) showed no difference when compared with sample times during a typical LD 12:12 lighting regimen where clear statistical differences existed. Data from fetal retinas showed no significant sustainable pattern in any of the measured variables. These quantitative findings have led to the conclusion that while alterations in perimeter measurements may be explained by using the vesicle recycling hypothesis, observed changes in terminal size and shape may be controlled by a light-initiated or light-enhanced mechanism and effected through an annular configuration of cross-striated fibrils found within these photoreceptor synaptic terminals.     

Ultrastructural changes in the neuropile of the sensorimotor cortex during long-term protein-calorie deficiency

The effect of alimentary (protein-caloric) deficiency on the brain sensomotor cortex of mice was studied during their postnatal development. It was found that malnutrition of mice from day 10 to day 40 of postnatal period brought about the most remarkable changes in synaptic junctions located on the dendritic spines. They manifested destruction of the spine apparatus, reduction of the width of synaptic slits and of postsynaptic membranes. Dystrophic and destructive alterations are frequently encountered in dendrites, axon terminals, and myelinized axons, indicating the reduced compensatory functions of the CNS.     

SMN requirement for synaptic vesicle, active zone and microtubule postnatal organization in motor nerve terminals

Low levels of the Survival Motor Neuron (SMN) protein produce Spinal Muscular Atrophy (SMA), a severe monogenetic disease in infants characterized by muscle weakness and impaired synaptic transmission. We report here severe structural and functional alterations in the organization of the organelles and the cytoskeleton of motor nerve terminals in a mouse model of SMA. The decrease in SMN levels resulted in the clustering of synaptic vesicles (SVs) and Active Zones (AZs), reduction in the size of the readily releasable pool (RRP), and the recycling pool (RP) of synaptic vesicles, a decrease in active mitochondria and limiting of neurofilament and microtubule maturation. We propose that SMN is essential for the normal postnatal maturation of motor nerve terminals and that SMN deficiency disrupts the presynaptic organization leading to neurodegeneration.     

Seasonal differences in the physiology and morphology of crayfish motor terminals

The physiology and morphology of identified crayfish motor terminals were compared at different seasons. We examined initial excitatory postsynaptic potential (EPSP) amplitudes, synaptic fatigue, and the frequency of synaptic varicosities along the motor terminals of an identified phasic motoneuron in animals collected over a period of 5 years. The physiology and morphology of identified crayfish motor terminals are different for animals collected in summer and winter. In winter animals, phasic axon motor terminals in the claw closer muscle produce large EPSPs initially, but show dramatic synaptic fatigue during repetitive stimulation. In summer animals, these terminals produce smaller initial EPSPs, but are more fatigue resistant. Due to their greater fatigue resistance, synaptic terminals have a greater over-all capacity for transmitter release in summer animals than do those of winter animals. Morphologically, terminals in summer animals have more synaptic varicosities, this result supports earlier studies that have shown that fatigue-resistant motor terminals have more synaptic varicosities. Experiments in which the electrical activity of the motoneuron was experimentally altered suggest that these differences in motor terminals may be due to seasonal differences in activity.     

Repeated exposure to methamphetamine causes long-lasting presynaptic corticostriatal depression that is renormalized with drug readministration

Addiction-associated behaviors such as drug craving and relapse are hypothesized to result from synaptic changes that persist long after withdrawal and are renormalized by drug reinstatement, although such chronic synaptic effects have not been identified. We report that exposure to the dopamine releaser methamphetamine for 10 days elicits a long-lasting (>4 month) depression at corticostriatal terminals that is reversed by methamphetamine readministration. Both methamphetamine-induced chronic presynaptic depression and the drug's selective renormalization in drug-experienced animals are independent of corresponding long-term changes in synaptic dopamine release but are due to alterations in D1 dopamine and cholinergic receptor systems. These mechanisms might provide a synaptic basis that underlies addiction and habit learning and their long-term maintenance.     

Properties of fast endocytosis at hippocampal synapses

Regulation of synaptic transmission is a widespread means for dynamic alterations in nervous system function. In several cases, this regulation targets vesicular recycling in presynaptic terminals and may result in substantial changes in efficiency of synaptic transmission. Traditionally, experimental accessibility of the synaptic vesicle cycle in central neuronal synapses has been largely limited to the exocytotic side, which can be monitored with electrophysiological responses to neurotransmitter release. Recently, physiological measurements on the endocytotic portion of the cycle have been made possible by the introduction of styryl dyes such as FM1-43 as fluorescent markers for recycling synaptic vesicles. Here we demonstrate the existence of fast endocytosis in hippocampal nerve terminals and derive its kinetics from fluorescence measurements using dyes with varying rates of membrane departitioning. The rapid mode of vesicular retrieval was greatly speeded by exposure to staurosporine or elevated extracellular calcium. The effective time-constant for retrieval can be < 2 seconds under appropriate conditions. Thus, hippocampal synapses capitalize on efficient mechanisms for endocytosis and their vesicular retrieval is subject to modulatory control.     

Agrin and Synaptic Laminin Are Required to Maintain Adult Neuromuscular Junctions

As synapses form and mature the synaptic partners produce organizing molecules that regulate each other’s differentiation and ensure precise apposition of pre- and post-synaptic specializations. At the skeletal neuromuscular junction (NMJ), these molecules include agrin, a nerve-derived organizer of postsynaptic differentiation, and synaptic laminins, muscle-derived organizers of presynaptic differentiation. Both become concentrated in the synaptic cleft as the NMJ develops and are retained in adulthood. Here, we used mutant mice to ask whether these organizers are also required for synaptic maintenance. Deletion of agrin from a subset of adult motor neurons resulted in the loss of acetylcholine receptors and other components of the postsynaptic apparatus and synaptic cleft. Nerve terminals also atrophied and eventually withdrew from muscle fibers. On the other hand, mice lacking the presynaptic organizer laminin-α4 retained most of the synaptic cleft components but exhibited synaptic alterations reminiscent of those observed in aged animals. Although we detected no marked decrease in laminin or agrin levels at aged NMJs, we observed alterations in the distribution and organization of these synaptic cleft components suggesting that such changes could contribute to age-related synaptic disassembly. Together, these results demonstrate that pre- and post-synaptic organizers actively function to maintain the structure and function of adult NMJs.     

Electron microscopic study on the innervation of the pancreas of the domestic fowl

Summary The innervation of the pancreas of the domestic fowl was studied electron microscopically. The extrapancreatic nerve is composed mostly of unmyelinated nerve fibers with a smaller component of myelinated nerve fibers. The latter are not found in the parenchyma. The pancreas contains ganglion cells in the interlobular connective tissue. The unmyelinated nerve fibers branch off along blood vessels. Their synaptic terminals contact with the exocrine and endocrine tissues. The synaptic terminals can be divided into four types based on a combination of three kinds of synaptic vesicles. Type I synaptic terminals contain only small clear vesicles about 600 Å in diameter. Type II terminals are characterized by small clear and large dense core vesicles 1,000 Å in diameter. Type III terminals contain small clear vesicles and small dense core vesicles 500 Å in diameter. Type IV terminals are characterized by small and large dense core vesicles. The exocrine tissue receives a richer nervous supply than the endocrine tissue. Type II and IV terminals are distributed in the acinus, and they contact A and D cells of the islets. B cells and pancreatic ducts are supplied mainly by Type II terminals, the blood vessels by Type IV terminals.This work was supported by a scientific research grant (No. 144017) and (No. 136031) from the Ministry of Education of Japan to Prof. M. Yasuda     

Alterations in the development of catecholamine turnover induced by perinatal methadone: Differences in central vs. peripheral sympathetic nervous systems

Administration of methadone to pregnant and nursing rats slows synaptogenesis of central cathcholaminergic systems in the offspring but accelerates the onset of synaptic function in peripheral sympathetic pathways. Norepinephrine turnover, assessed by inhibiting catecholamine biosynthesis with alpha-methyl-p-tyrosine, was elevated in cardiac sympathetic nerve terminals in rats exposed perinatally to methadone. In contrast, turnover was unchanged in noradrenergic and dopaminergic systems in the brain. Similar results were obtained when methadone was given directly to the pups during postnatal life. These data suggest that opiate-induced alterations of impulse flow and transmitter turnover in a given neuron population may determine whether the effects of perinatal methadone exposure result in facilitation or inhibition of synaptic development.     

应用电镜X射线显微分析方法研究神经细胞内钙离子的分布

本文应用X射线能谱分析结合电镜技术研究了钙离子在青蛙交感神经节神经元内的分布及其在茶碱作用下分布的变化.实验结果表明在组织样品的电子致密沉积物EDD中含有钙离子成分.在青蛙交感神经节突触后神经元中,包含钙离子的EDD存在于质膜、亚表面池及线粒体中;在突触前神经末梢中,突触小泡的膜上也可观察到EDD.在茶碱作用下,交感神经节神经元的质膜、线粒体中的EDD大大地减少;在亚表面池中则没有或很少观察到EDD;突触前末梢中的突触小泡明显地趋向聚集,在突触小泡之间的连接处频繁地出现EDD.本文根据实验结果讨论了茶碱可能促使钙离子从交感神经元的上述部位中释放出来,并认为质膜、亚表面池和线粒体是细胞内钙离子的贮存部位,而亚表面池可能是主要的贮存释放部位.突触前神经末梢内形态上的变化可能与神经递质释放的机理有关.     

SYNAPTIC VESICLE DEPLETION AND RECOVERY IN CAT SYMPATHETIC GANGLIA ELECTRICALLY STIMULATED IN VIVO : Evidence for Transmitter Secretion by Exocytosis

This study examined the ultrastructure of presynaptic terminals after short periods of vigorous acetylcholine (ACh) secretion in the cat superior cervical ganglion in vivo. Experimental trunks of cats anesthetized with chloralose-urethane were stimulated supra-maximally for periods of 15–30 min and at several frequencies including the upper physiological range (5–10 Hz). Stimulated and contralateral control ganglia from each animal were fixed by intra-arterial aldehyde perfusion, processed simultaneously, and compared by electron microscopy. Stimulation produced an absolute decrease in the number of synaptic vesicles, an enlargement of axonal surface membrane, and distinct alterations in the shape of presynaptic terminals. Virtually complete recovery occurred within 1 h after stimulation at 10 Hz for 30 min. These results support the hypothesis that ACh release at mammalian axodendritic synapses occurs by exocytosis of synaptic vesicles resulting in the incorporation of vesicle membrane into the presynaptic membrane and that synaptic vesicles subsequently are reformed from plasma membrane.     

Interneuronal synapses in the local ganglia of the cat urinary bladder.

The interneuronal synapses of the urinary bladder in the cat were studied by electron microscopy. The great majority of the fibres containing vesicles are found within the ganglia occurring in the trigonum area. Morphologically differentiated synaptic contacts could be observed on the surface of the local neurons and between the different nerve processes. The presynaptic terminals can be divided into three types based on a combination of synaptic vesicles. Type I terminals, presumably cholinergic synaptic terminals, contain only small clear vesicles of 40-50 nm in diameter. Type II terminals, presumably adrenergic terminals, are characterized by small granulated vesicles of 40-60 nm in diameter. Type III terminals, probably of local origin, contain a variable number of large granulated vesicles of 80-140 nm in diameter. Occasionally, a single nerve fibre contacted several (two or four) other nerve processes forming a typical synapse. In other cases, on one nerve cell soma or on other nerve processes there are two or three different-type nerve terminals establishing synapses. It might be inferred from these observations that convergence and divergence can occur in the local ganglia and that cholinergic and adrenergic synaptic terminals can modulate the ganglionic activity. However, a local circuit also can play an important role in coordinating the function of the bladder.     

Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber

Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.     

A retinal projection to the lateral hypothalamus in the rat

Summary This study presents evidence for a retinal projection to neurons in the lateral hypothalamic area (LHA) of the albino rat. In Golgi-Kopsch material dendrites from LHA-neurons are observed to extend through the supraoptic commissures into the optic tract. The presence of dendrites in the optic tract is confirmed by electron microscopy. Numerous axon terminals are observed forming asymmetric synaptic contacts with these dendritic profiles. Following bilateral enucleation, many of the preterminal axons and terminals in synaptic contact with dendrites in the optic tract demonstrate dark degeneration. After intraocular injection of horseradish peroxidase, there is marked labeling of preterminal axons and terminals in the optic tract. These observations indicate that LHA neurons receive a direct retinal projection from terminals making synaptic contact with dendrites of LHA-neurons extending into the optic tract.     

Synaptic organization of the cat parietal association cortex (area 5b)

An electron microscopy study was made of synaptic organization in the cat association cortex, area 5b. A total of 1635 axonal terminals were discovered over 6215 µm² (240 electronic imagings of slices of different association cortex layers); i.e., an average of 263±16 terminals per 1000 µm² expanse. It was found that 75.5% of axon terminals contained synaptic vesicles and formed either one- or two-sided contact with postsynaptic structures; 24.5% of axonal terminals contained synaptic vesicles but formed no distinct synaptic contacts with nearby neurons; 84.9% of terminals contained round-shaped or slightly oval synaptic vesicles; 7.8% had both rounded and elongated shapes, and vesicles were very elongated in the remaining 7.3%. Of the axonal terminals having synaptic contacts, axo(dendritic)-spinal terminals accounted for 46.6%, and axodendritic and axosomatic endings amounted to 50.0% and 3.4% respectively (in all 77% of axosomatic terminals contained elongated vesicles and maintained symmetrical contact, while 23% had round-shaped vesicles and formed asymmetrical contact). Calculations show that for each 1 mm³ an average of 258 million axonal terminals are found forming synaptic contacts in the cat association cortex as well as 84 million terminals containing synaptic vesicles but not forming contact.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 174–185, March–April, 1989.     

The colocalization of neurotransmitters in the presynaptic boutons of inhibitory synapses in the lamprey spinal cord]

Using the electron microscopy immunocytochemistry, the GABA and glycine immunoreactivity was studied in presynaptic axon terminals of the spinal cord central gray in the lamprey Lampetra fluviatilis. All immunopositive presynaptic terminals contacting motoneurones or non-identified post-synaptic profiles were divided into only GABA- (44%), only glycine-immunopositive terminals (26%), and both GABA- and glycine-containing terminals (30%). The glycine-immunopositive axon terminals contained flattened synaptic vesicles. Large dense core vesicles were co-localised with conventional synaptic vesicles in 74% of GABA-containing presynaptic terminals.     

Localization of substance P and leucine enkephalin in the nerve terminals of the guinea pig paracervical ganglion

Summary Immunoreactivities (IR) of substance P and leucine enkephalin have been demonstrated in the guinea-pig paracervical ganglion by an immunogold electron microscope method. Both substance P-IR and leucine enkephalin-IR were detected in large synaptic vesicles with electron-dense cores. The former neuropeptide was detected in nerve terminals and varicosities comprised mainly of large vesicles with electron-dense cores; the latter was detected in nerve terminals and varicosities that also included small, clear synaptic vesicles. In a minority of nerve terminals and varicosities coexistence of both immunoreactivities could be demonstrated within vesicles with an electron-dense core. Also present in these nerve terminals and varicosities were small, clear synaptic vesicles, though these were unreactive.