Numerous Varicosities Research Articles

Fine structure of the synaptic endings between sympathetic axons and skeletal muscle cells and of the varicosities in the bundles of neurites in tissue culture

In cocultures of skeletal muscle and sympathetic ganglia from chick embryos, synaptic boutons on skeletal muscle cells and varicosities in the neuritic bundles were observed electron microscopically. Synaptic endings on skeletal muscles were bulbous. Most synaptic boutons simply made contact with muscle fibers, but some boutons appeared as concave invaginations into the sarcolemma. In the neuritic bundles, numerous varicosities were observed. Close approximations were found between the synaptic boutons and skeletal muscles and between the varicosities and neurites (dendrites or axons), but no membrane thickening nor subsynaptic infolding was observed in either synapse; in our cultures the contacts were characteristic of the autonomic nervous system. In both synapses, three variations were demonstrated by KMnO 4 fixation after 5-hydroxydopamine incubation: (i) containing predominantly small dense-core vesicles (noradrenergic type), (ii) predominantly small clear vesicles (cholinergic types), and (iii) a mixture of both small dense-core and small clear vesicles (mixed type). Cytochemically, the varicosities in the neuritic bundles were predominantly noradrenergic at 1 week in culture, and both the synaptic boutons on skeletal muscles and the varicosities in the neuritic bundles contained a mixed population of small dense-core and small clear vesicles at intermediate times, with a gradual shift to cholinergic characteristics. These findings strongly suggest that in this culture system some sympathetic neurons or fibers become cholinergic (neuronal plasticity).

Evidence for neurotransmitter plasticity in vivo: Developmental changes in properties of cholinergic sympathetic neurons

We have examined the cholinergic sympathetic innervation of sweat glands in footpads of adult and developing rats. Acetylcholinesterase staining reveals a plexus of heavily stained fibers in the sweat glands of adult rats. Reaction product appears among and around bundles of axons that lie at a considerable distance from the cells of the secretory tubule. Each bundle contains 8–12 axons that possess numerous varicosities and contain small clear and large dense core vesicles. The glands of the hindpaws and their innervation develop during the first three weeks after birth. Catecholamine-containing axons were associated with the forming glands. At 7 and 10 days, intensely fluorescent fibers surrounded the tubules, and all of the axon profiles associated with the glands contained small granular vesicles (SGV) after permanganate fixation to reveal vesicular stores of norepinephrine. At 14 days the sweat gland plexus was less intensely fluorescent than at earlier ages and relatively few SGV were present. By 21 days, no endogenous catecholamine fluorescence and no SGV were detectable. However, following exposure to exogenous catecholamine, fluorescent fibers were present in the sweat glands of mature rats and they corresponded in position and density to the plexus localized with acetylcholinesterase staining. Catecholamine uptake was blocked by incubation in the cold and by desmethylimipramine and was not observed in cholinergic parasympathetic fibers in the iris or salivary glands. After intraperitoneal administration of 5-hydroxydopamine and permanganate fixation, all the axons in the sweat glands contained a few SGV. Thus, the developing sweat glands appear to be innervated by noradrenergic axons that lose their stores of endogenous catecholamines but not their capacity for uptake and storage as they elaborte an axonal plexus in the maturing glands. These observations support the hypothesis that cholinergic sympathetic neurons appear to undergo a transition from noradrenergic to cholinergic function during development in vivo similar to that previously described in cell culture.

Identified nonspiking local interneurons mediate nonrecurrent, lateral inhibition of crayfish mechanosensory interneurons

1. We have used intracellular neuropil recordings and dye injection to identify a pair of nonspiking, Local, Directionally Selective (LDS) interneurons in the terminal ganglion of the crayfish,Procambarus clarkii. The neurons have bilateral but asymmetric processes connected by a thick (ca. 17 μm) segment in the dorsal neuropil. The processes ipsilateral to the soma are smooth while those on the other side have numerous varicosities (Figs. 1, 2). 2. We found headward water movement to be the best natural stimulus for depolarizing the LDS interneurons (Fig. 3). Neither water movement nor any combination of electrical stimulation of afferents and current injection into the LDS interneurons ever caused them to spike. 3. The receptive field of the LDS interneuron is mainly restricted to the uropods ipsilateral to its cell body, and within that field the afferents from the endopodite provide the strongest excitation. The afferent-produced PSPs are chemically mediated and are probably monosynaptic; they occur with an estimated synaptic delay of approximately 1 ms (Figs. 4, 5). 4. The output of the LDS interneuron is contralateral to the input side and is associated with the processes having varicosities. Thus, although the neuron is nonspiking, it is functionally polarized to receive input on one side of the ganglion and convey it to the other side. 5. All known output connections of the LDS interneuron are inhibitory and highly specific. The LDS interneuron exclusively inhibits projection interneurons excited by headward afferents on the output side. Not all headward projection interneurons are inhibited equally (Fig. 6 and Table 1). 6. The inhibitory effect of the LDS interneuron is mediated postsynaptically by chemical IPSPs (Fig. 7). The LDS cell does not cause inhibition at rest potential, and the threshold for causing inhibition in at least one postsynaptic interneuron was 10 mV of depolarization of the LDS cell. The inhibitory pathway between the LDS interneuron and the postsynaptic neurons is stable (Figs. 8, 9). 7. The LDS interneurons mediate lateral inhibition that is both nonrecurrent and reciprocal. Because the LDS interneurons are nonspiking, once above threshold they deliver a precise but inverted copy of the afference on one side to the projecting interneurons on the other side. These features have many consequences, including the ability to attenuate common signals that arise from ambient water movement, and conversely to accentuate the different afference produced by laterally asymmetric signals.

Radioautographic study of the symphathetic fibers in the cochlea.

The noradrenergic fibers of the cochlea have been investigated by radioautography after an in vitro labeling by 3H-Noradrenalin (3H-NA). Unmyelinated, perivascular and non-perivascular fibers took up and retained 3H-NA. Non-perivascular fibers formed small bundles together with unmyelinated efferents in the intraganglionic spiral bundle and the osseous spiral lamina. No labeled fibers were detectable beyond the habenula inside the organ of Corti. The labeled fibers, which are probably noradrenergic, were characterized by numerous varicosities and discontinuous Schwann sheathes. These morphological characteristics could associate them with a general role in cochlear function. Sometimes, labeled fibers were also found very close to Ranvier's node of certain myelinated fibers, which could suggest a specific effect on these fibers.

Dopaminergic amacrine cells in the cat retina

Autoradiographic studies of cat retina showed an accumulation of [ 3H]dopamine in a subpopulation of amacrine cells whose process ramify in the outermost stratum of the inner plexiform layer. Dendrites of these cells are characterized by numerous varicosities measuring up to 2 μm in diameter which are connected by fine intervaricose segments. The dopaminergic amacrine cells are presynaptic to other labeled cells and to unlabeled amacrine populations but not to bipolar or ganglion cells. [ 3H]Dopamine-labeled processes provide extensive synaptic input to the somata and lobular appendages of type AII amacrine cells. This relationship suggests that dopaminergic amacrine cells may play an important role in the regulation of rod pathways in the cat retina.

The innervation pattern of crustacean skeletal muscle. Morphometry and ultrastructure of terminals and synapses.

The innervation pattern of distal muscle fibers of the opener muscle of walking legs of crayfish (Astacus leptodactylus) was investigated using methylene-blue staining, cobalt infiltration, and electron microscopy. A quantitative analysis of the entire innervation of single muscle fibers was attempted. It was found that instead of the generally assumed parallel array of numerous excitatory and inhibitory terminals, innervation consists of only a few branched terminals. The branches of excitatory and inhibitory terminals lie side-by-side. Both types are characterized by numerous varicosities (see Fig. 9B). The aggregate length of excitatory as well as inhibitory terminals on one muscle fiber is, on the average, about 1,500 micrometer with a total of 152 varicosities spaced about 10 micrometer apart. The average diameter of the varicosities is 4.26 micrometer, that of the connecting thin segments about 0.5 micrometer. Total terminal surface of motor or inhibitory terminals amounts to about 10,000 micrometers2 per muscle fiber. There are approximately 2,000 motor synapses on each muscle fiber, but their average total area is only about 6% of the terminal membrane area, or 0.06% of the (idealized) muscle fiber surface. There are conspicuous differences in the postsynaptic specializations associated with excitatory and inhibitory terminals; these are described in detail. The results are discussed in a functional context and with regard to design and results of electrophysiological experiments.

Somatostatin: Localization and distribution in the cortex and the subcortical white matter of human brain

Examination of the cortex and the subcortical white matter by use of an immunocytochemical technique—the per oxidase anti-per oxidase method—shows that somatostatin is located in a wide-spread neuron system with cell bodies localized in both the cortex and the subcortical white matter of the human brain. In the cortex, the somatostatin cell bodies and fibers are found in all layers, but the fibers are especially numerous in layer I located tangentially to the brain surface. The fibers are very long and subdivide into many branches which form a network of pathways in the deeper cortical layers. There are numerous varicosities along the fibers and they come into close contact with other non-immunoreactive neuronal cells. The somatostatin cells located in the white matter are larger than the somatostatin cells in gray matter. They are giant cells with a size ranging from 50 to 120 μm. The fibers from these cells are varicose and can be followed both rostrally into the cortical gray matter and caudally in the subcortical white matter. The localization and the morphology of the somatostatin neurons in the cortex and the subcortical white matter indicate that somatostatin may be able to exert sustained influence in various brain areas and thereby modulate integrative and/or specific functions, not only via connection in the gray matter but also by influencing the neuronal circuits passing through the subcortical white matter.

Immunohistochemical studies on the serotonergic innervation of the pia mater.

The direct innervation of the pial blood vessels by serotonin neurons has been demonstrated with a modified peroxidase-antiperoxidase technique in the mammalian central nervous system. The pia mater covering the ventrolateral surface of the medulla oblongata is innervated by numerous varicose serotonin fibers originating from the serotonin neurons of the lower brainstem. Scattered serotonin fibers were observed in the pia mater in every part of the brain and spinal cord.

Fate of presynaptic afferents to Purkinje cells in the adult nervous mutant mouse: A model to study presynaptic stabilization

The hemispheric cerebellar cortex from 174- to 420-day-old nervous mice has been studied with Golgi, Cajal-reduced silver and electron microscopic techniques. In all mice, the existence of a continuous process of Purkinje cell death, indicated by the presence of few of these neurons in early stages of degeneration, has been established. The morphology of such degenerating cells, as well as the removal of the necrotic debris and the consecutive isolation of presynaptic fibers by glia are described. In the molecular layer, and in spite of the important Purkinje cell loss, numerous varicosities belonging to parallel fibers persist. They can be either synapsing on interneurons or concentrated in small clusters of 2–10 boutons covered by astrocytic processes. The synaptic investment of stellate and basket cells, as well as the length and the branching pattern of their dendritic fields are not significantly changed, besides the increase in afferent terminals deprived of their postsynaptic targets. Basket formations, as numerous as in control cerebellum, are forming a discontinuous row at the superficial third of the granular layer. Over 90% of these baskets are devoid of Purkinje cells, however, their ultrastructural features mimic those of the same terminals in control mice, with the exception that few of them can either establish heterologous synapses with granule cell dendrites, or develop gap junctions between them. The significance of the persistence of normal looking presynaptic elements long time after deprvation of their postsynaptic targets is discussed in relation to the role that function can play in the stabilization of synaptic contacts.

Effects of chronic deafferentation on adrenergic ganglion cells and small intensely fluorescent cells.

To determine the reaction of adrenergic ganglion cells and small intensely fluorescent (SIF) cells to chronic deafferentation, catecholamine fluorescence of the major pelvic ganglion (MPG) of the rat has been studied following section of the hypogastric nerve, pelvic nerve and sympathetic trunk. Only minor changes occurred following section of the hypogastric nerve; the fluorescence surrounding a few adrenergic ganglion cells became brighter. In contrast, pelvic neurectomy resulted in the appearance of numerous varicose fibres and an increase in the fluorescent intensity of fibres enclosing many ganglion cells. Varicose fibres seem to originate from adrenergic ganglion cells and SIF cells. In many instances, nests of SIF cells gave rise to radially oriented fibres. Removal of the sympathetic trunk appeared to have no effect on the MPG. It is suggested that the appearance of varicose fibres from SIF cells following deafferentation may be due to collateral sprouting of these cells or to the increased fluorescence of pre-existing processes.

Consecutive demonstration of nerves containing catecholamine and acetylcholinesterase in the rat cornea.

The innervation of the cornea of newborn (two day old) and adult rats was investigated using glyoxylic-acid-induced fluorescence (GIF) for catacholamines and subsequent acetylcholinesterase reaction. Fluorescent nerve were observed around the limbal vessels and in the pericorneal nerve plexus, from which they branched towards the central parts of the cornea. The fluorescent corneal nerves were either nonvaricose or had varicosities at intervals of 10 micra. When the animals had been pretreated with nialamide, noradrenaline and propranolol, some fluorescent branching nerve terminals with numerous varicosities also appeared. All fluorescent nerves disappeared two days after ipsilateral superior cervical sympathectomy. When the acetylcholinesterase (AChE) reaction was performed subsequently to the GIF reaction the following nerve types could be identified: 1. nerves containing both catecholamine (CA) fluorescence and AChE, 2. Nerves containing only AChE.

Mode of distribution of aminergic fibers in the cerebellar cortex of the chicken

The cerebellar cortex of adult hens contains a dense plexus of thin varicose nerve fibers which display a formaldehyde-induced green fluorescence. This plexus is not distributed at random in the cortical layers. Within the granular layer the plexus forms a netlike pattern. The fiber branches, which have numerous varicosities, are predominantly oriented in the traverse plane of the folium. In the molecular layer the fluorescent plexus shows some variations in the convex, flat and concave portions of the folia. Many of the fluorescent branches are oriented parallel to the course of the folium. They arise from a T-division of radially oriented axons resembling parallel fibers in Golgi sections. The meshes of the fluorescent plexus in the granular layer measure 10-60 mu. In the molecular layer (top of the folia) there are about 30 fluorescent fibers per 100 mu2. The fluorescent fibers originate from the locus coeruleus and form a rostral and a caudal bundle in the cerebellar peduncle. The mode of distribution of the fluorescent fibers in the cortical layers seems to depend on the organization of the innervated tissue. Light microscopy suggests that the aminergic fibers innervate more than one class of cerebellar neurons.

Ultrastructural organization of the sensory fibers innervating the Golgi tendon organ.

AbstractThe organization of the sensory innervation of Golgi tendon organs (GTO) from E. carpi ulnaris of the cat is described. The large Ib afferent fiber gives rise to small myelinated branches which become oriented longitudinally in a neural compartment of the GTO capsule lumen. The latter give rise to smaller, unmyelinated branches which further arborize among loosely packed collagen bundles. The unmyelinated fibers form numerous varicosities and small terminal branches which have portions of the axolemma covered only by basal lamina which intervenes between the collagen bundles and the axolemma. In places, the axon's surface may lack all investments and nerve and collagen come into intimate association. The large unmyelinated branches and their varicosities have different axoplasmic characteristics compared with the small unmyelinated terminal portions. The latter contain few or no mitochondria, vesicles, neurotubules, and neurofilaments. In addition to the description of the terminals, the similarities and differences of the innervation of the GTO, compared with other mechanoreceptors, are discussed.

Differences in the distribution of catecholamine varicosities in cat and rat reticular formation.

The distribution of catecholamine varicosities within the brainstem reticular formation of the immature cat was determined by means of the formaldehyde-induced fluorescence technique. A continuous pattern of intense, green, medium-sized varicosities exists at nearly all brainstem levels. At most of these levels the varicosities appear within the boundaries of reticular formation nuclei. However, in rostral mesencephalon, some of the varicosities of the pattern lie in proximity to perikarya of the red nucleus. In addition, numerous varicosities in caudal medulla appear to extend from the pattern into nonreticular formation nuclei. A comparable pattern of reticular formation fluorescence is absent in the rat and this finding is believed to represent a true interspecies difference.

Visual identification of synaptic boutons on living ganglion cells and of varicosities in postganglionic axons in the heart of the frog.

1. Parasympathetic neurons were studied in the transparent interatrial septum of the frog (Rana pipiens) with light- and electron-microscopic techniques. The aim was to identify visually cellular and subcellular details in a living preparation, especially synaptic boutons on ganglion cells and the varicosities in postganglionic axons supplying the muscles of the heart. 2. The interatrial septum contains the following nervous elements: unipolar parasympathetic ganglion cells, their preganglionic vagal innervation, postganglionic sympathetic axons and sensory fibres. These structures and the nuclei of their related Schwann cells can be viewed with various optical systems, especially differential interference contrast optics. The same neural elements identified in the live preparation can be sectioned for electron microscopy. 3. Most ganglion cells are innervated by a single presynaptic axon, terminating in up to 27 synaptic boutons which on the average cover about 3.0 % of the surface of nerve cell bodies. A few scattered boutons also occur on the initial axonal portion of the ganglion cells. 4. Synaptic boutons on ganglion cells were recognized in the living unstained preparation. Their identity was confirmed by electron microscopy and by light microscopy combined with methylene blue, zinc iodide and osmium, and cholinesterase staining methods. 5. The terminal branches of postganglionic axons have numerous varicosities along their course. Some are as close as a few hundred angstroms (10 Å = 1 nm) to muscle fibres, others are many pm away. There are two types of varicosities: (i) those which contain predominantly granular vesicles characteristic of neurons releasing catecholamines, and (ii) those with predominantly agranular vesicles which belong to the cholinergic axons of septal ganglion cells. Regardless of their distance from muscle fibres, the cholinergic varicosities have the same fine structural features, including membrane thickenings, as synaptic boutons on the ganglion cells. These findings support earlier suggestions that the varicosities along postganglionic axons are a series of transmitter release sites. 6. Varicosities were observed in the live septum; their identity was confirmed by subsequent electron microscopy. Many live varicose axons were traced back to the vicinity of individual septal ganglion cells. Additional evidence that they belonged to a particular ganglion cell, and were therefore cholinergic, was obtained by injecting Procion yellow into the cell body and observing the neuron with a fluorescence microscope after the dye had spread into the axonal processes. Time lapse photography of up to 24 h showed no ‘ peristaltic ’ movement of varicosities. 7. Granular or agranular vesicles also occur along cylindrical axons within nerve bundles many pm away from muscle fibres. Like the vesicles in varicosities, they are clustered close to ‘thickenings’ in the surface membrane and belong to postganglionic nerve fibres. 8. Ganglion cells in isolated septa survive for 2 weeks or longer, still giving membrane potentials and impulses. Time lapse cinematography for up to 2 weeks after removing the septum showed that the organelles within the neurons were in motion and that a two-way traffic takes place between the cell body and axon, as commonly found in cultured neurons.