Innervation of mouse lymph nodes: nerve endings on muscular vessels and reticular cells

Lymph node nerve endings have been studied in 1- to 48-day-old mice. Serial sections of Epon-embedded lymph nodes were observed under the electron microscope to find the nerve endings. Most lymph node nerve fibers finally reach the smooth muscle cells of arterioles and muscular venules. Both kinds of vascular endings are similar, although endings are less numerous on venules. Nerve endings consist of one or more nerve processes surrounded by a usually incomplete Schwann cell sheath; frequently, axons show wide areas directly facing the muscle cells. The distance between such a naked axon and a myocyte ranges from 100 to 800 nm. Small granulated and clear vesicles are especially abundant in varicosities of nerve processes that are located very close to muscle cells. Nerve endings of lymph node vasculature probably correspond to vasomotor sympathetic adrenergic endings, regulating the degree of contraction of vessels which have a muscular layer. Other kinds of nerve endings also exist in lymph nodes: some axons appear free in the stroma and contact the surfaces of reticular cells; the latter also extend delicate cytoplasmic processes that surround the axons. The functional significance of nerve cell-reticular cell contacts is unknown.     

Light- and electron-microscopic study of synaptic connections in the paracervical ganglion of the female rat: special reference to calcitonin gene-related peptide-, galanin- and tachykinin (substance P and neurokinin A)-immunoreactive nerve fibers and terminals

Nerve fibers and varicosities in the pelvic paracervical ganglia (PG) are immunoreactive for the neuropeptides calcitonin gene-related peptide, galanin, and the tachykinins substance P and neurokinin A. Many of these fibers and varicosities are capsaicin-sensitive, originate in dorsal root ganglia and, thus, are considered to be primary afferent fibers. Numerous immunoreactive varicosities are pericellular to principal neurons in the PG. The present study examines the ultrastructure of calcitonin gene-related peptide-, galanin-, substance P-, and neurokinin A-immunoreactive nerve fibers and varicosities in the ganglia to determine their relationships to principal neurons and their synaptic connectivity. Paracervical ganglia of female rats were processed for light-microscopic immunohistochemistry using antisera against synapsin I, as a nerve terminal marker, and microtubule-associated protein-2 to define soma and dendrites. The rationale for performing this co-immunohistochemical analysis was to reveal the relationship between nerve endings and principal neurons. Synapsin I endings were predominantly axosomatic with fewer being axodendritic. Other ganglia were processed for electron-microscopic immunohistochemistry using both standard immunogold and peroxidase-anti-peroxidase procedures. Unmyelinated fibers and varicosities immunoreactive for calcitonin gene-related peptide, galanin, and the tachykinins were routinely observed in the interstitium between neuron somas. Numerous immunoreactive axon profiles were present in small groups that were ensheathed by Schwann cells. Immunoreactive fibers and varicosities were also observed within the satellite-cell sheath of the neuron soma and often intimately associated with the membrane of the soma, somal protrusions, or with the proximal part of a dendrite. Membrane specializations, indicative of synaptic contacts, between the fibers and the principal neurons were observed. It is suggested that these peptide-immunoreactive sensory fibers and varicosities are involved in regulation of activity in the PG.     

Bergmann glia and the recognition molecule CHL1 organize GABAergic axons and direct innervation of Purkinje cell dendrites

The geometric and subcellular organization of axon arbors distributes and regulates electrical signaling in neurons and networks, but the underlying mechanisms have remained elusive. In rodent cerebellar cortex, stellate interneurons elaborate characteristic axon arbors that selectively innervate Purkinje cell dendrites and likely regulate dendritic integration. We used GFP BAC transgenic reporter mice to examine the cellular processes and molecular mechanisms underlying the development of stellate cell axons and their innervation pattern. We show that stellate axons are organized and guided towards Purkinje cell dendrites by an intermediate scaffold of Bergmann glial (BG) fibers. The L1 family immunoglobulin protein Close Homologue of L1 (CHL1) is localized to apical BG fibers and stellate cells during the development of stellate axon arbors. In the absence of CHL1, stellate axons deviate from BG fibers and show aberrant branching and orientation. Furthermore, synapse formation between aberrant stellate axons and Purkinje dendrites is reduced and cannot be maintained, leading to progressive atrophy of axon terminals. These results establish BG fibers as a guiding scaffold and CHL1 a molecular signal in the organization of stellate axon arbors and in directing their dendritic innervation.     

Electron microscopy of the arcuate nucleus of normal and 5-hydroxydopamine treated cats

The arcuate nucleus of normal cats and of cats treated with 5-hydroxydopamine (5-OHDA) was investigated by electron microscopy. The neurons of the arcuate nucleus were classified into three types, clear, intermediate and dark, according to their fine structure. The clear type contained numerous dense-cored vesicles and well developed cell organelles. All three types were frequently seen to be partially surrounded by glial processes. Many axo-somatic and axo-dendritic synapses mostly small in diameter were also observed around the neurons. Synaptic contacts were demonstrated between axon endings and axonal processes which contained elementary granules. After administration of 5-OHDA small and large dense-cored vesicles appeared in the nerve endings surrounding the neurons. The relationship between the dense-cored vesicles in the perikarya and dopamine was briefly discussed.     

The accessory hyperstriatum of the domestic fowl

Summary The accessory hyperstriatum of normal domestic fowl (Gallus domesticus) was fixed with aldehydes followed by osmication and studied by electron microscopy. The relationships among neurons, neuroglia, and their processes is reported. Large and smaller neurons, astrocytes, and oligodendroglia are identified and their fine structure described. Most axonal endings contain spheroidal presynaptic vesicles, but a few terminals with flattened vesicles also are seen. Symmetrical and asymmetrical synaptic specializations of axon terminals are observed.     

Rapid arrest of axon elongation by brefeldin A: a role for the small GTP-binding protein ARF in neuronal growth cones

Members of the ADP-ribosylation factor (ARF) family of small guanosine triphosphate-binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings.     

The fine structure of the nerve cord of Myxicola infundibulum (annelida,polychaeta)

Summary Ultrastructural observations of the giant axon of Myxicola infundibulum reveal that the axoplasm contains neurofilaments, a few neurotubules and mitochondria. Finger-like projections issuing from the glial cells of the sheath encircle the giant axon at various angles. The space between the axolemma and sheath is 125 Å. Branches of the giant axon are also surrounded by a glial sheath as they course through the neuropil. Some branches of the giant axon seem to fuse with certain neurons, creating a syncytial arrangement between the giant axon and these neurons.Many small nerve fibers course longitudinally in the neuropil of the nerve cord. Most of these axons are separated from each other by a space of 200 Å without intervening glial processes. Synapses in the neuropil have both clear 600 Å vesicles and larger dense core vesicles suggesting chemical transmission. Some, but not all, of the synaptic areas show thickened membranes and dense material in the synaptic cleft.This study was supported in part by PHS NS-07740 to R.L.P., J.A.B. is a NDEA Predoctoral Fellow in the Department of Physiology.     

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing¹ as well as in pathological processes such as neuropathic pain². Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.     

Inductive role of mossy fibers of hippocampus in the development of dendritic spines in aberrant synaptogenesis at neurotransplantation

The dentate fascia of the hippocampal formation isolated from 20-day-old Wistar rat fetuses was subjected to heterotopic transplantation into the somatosensory area of the neocortex of adult rats of the same strain. Five months after surgery, neurotransplantates, together with neighboring area of the neocortex, were studied using light and electron microscopy. We carried out a detailed study of the ultrastructure of the ectopic synaptic endings formed by the axons of granular neurons of the dentate fascia (mossy fibers) with neurons of the neocortex unusual for them in a normal state. Ultrastructural analysis revealed that most ectopic synaptic endings produce its determinant morphological features: giant sizes of presynaptic knobs, active zones with branched dendritic spines, and adherens junctions with the surface of dendrites. The data indicate that the mossy fibers growing from neurotransplantates induce structural and chemical reorganization of dendrites of the neocortex using transmembrane adherens junctions, such as puncta adherentia junctions. This results in the differentiation of active zones and development of dendritic spines typical for giant synaptic endings that are invaginated into presynaptic endings. Thus, the ability of neurons of the dentate fascia to form aberrant synaptic connections at transplantation results from the inductive synaptogenic properties of mossy fibers.     

Ruffini endings are absent from the periodontal ligament of trkB knockout mice

To clarify the role of neurotrophin receptors in the development of Ruffini endings, periodontal ligaments and trigeminal ganglia of trkA, trkB, and trkC knockout mice were immunostained for protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), parvalbumin (PV), and calretinin (CR). Innervation patterns of PGP 9.5- and CGRP-immunoreactive fibers were examined in the periodontal ligament of the knockout mice. PGP 9.5-positive fibers in the incisal periodontal ligaments of trkA and trkC knockout mice form Ruffini endings distinguished by dendritic ramifications and branches. However, Ruffini endings were not present in the periodontal ligament of trkB knockout mice. Only free nerve endings were observed in tissue of trkB knockout mice. Compared with trkA and trkC knockouts, the proportion of CR-positive neurons in mandibular and maxillary regions of the trigeminal ganglion of trkB knockout mice is decreased. These findings indicate that the development of periodontal Ruffini endings is regulated by trkB-dependent and CR-coexpressing neurons.     

Histological study of motor innervation to long nuclear chain intrafusal fibers in the muscle spindle of the cat

Several muscle spindles of the cat tenuissimus muscle were cut in serial, 1-micron thick transverse sections and stained with toluidine blue in search for long nuclear chain intrafusal muscle fibers. Five complete poles of the long chain fibers were located. Each fiber pole displayed one plate-type motor ending situated in the extracapsular fiber region. The endings were supplied by myelinated motor axons that originated from intramuscular nerve fascicles containing motor axons to extrafusal muscle fibers. One of the endings was innervated by a collateral from a motor axon that supplied an extrafusal end-plate. Ultrastructurally, the long chain endings resembled extrafusal end-plates. They were more complex, in terms of prominence of sole-plate and degree of post-junctional folding, than any other intrafusal ending present in the spindles. The motor endings of the long chain fibers were assumed to be the terminals of static (fast) skeletofusimotor axons, which preferentially innervate the longest nuclear chain fibers of cat muscle spindles.     

Morphology and morphometry of motor endings on macaque intrafusal fibers

The ultrastructural studies have shown three types of motor endings in the macaque intrafusal fibers: 1) unindented axon terminals with smooth or shallowly folded postsynaptic membrane; 2) indented terminals with few postsynaptic folds; and 3) indented terminals with heavily folded postsynaptic membrane. The terminals on bag 1 and chain fibers were generally more indented than those on the bag 2 fibers. Deeply indented terminals with highly folded postsynaptic membranes were noticed on the bag 1 and chain endings in spindles from lumbrical but not the biceps muscle. In the individual intrafusal fibers from the biceps and lumbrical spindles, the degree of indentation did not correlate with the extent of postsynaptic folding (P greater than .01). Endings on bag 1 and chain fibers in the lumbrical spindles showed a positive correlation between indentation of terminals and their distance from the primary sensory endings (P less than .01), whereas the lumbrical bag 2 endings and the biceps intrafusal endings did not (P greater than .01). The shape of the intrafusal motor endings thus is independent of their location but dependent on the type of intrafusal fibers.     

Rapid arrest of axon elongation by brefeldin A: A role for the small GTP‐binding protein ARF in neuronal growth cones

Members of the ADP‐ribosylation factor (ARF) family of small guanosine triphosphate–binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 105–115, 1999     

Factors that determine the form of neuromuscular junctions of intrafusal fibers in the cat

The form of terminations of fusimotor (gamma) and skeletofusimotor (beta) axons on intrafusal fibers was analyzed in serial sections of 20 spindles of the cat tenuissimus muscle. Seven synaptic features were assessed either qualitatively or quantitatively from electron micrographs of transverse sections of 184 intrafusal and 30 extrafusal endings. Features were compared among endings that were terminations of gamma or beta axons on different types of intrafusal fiber at different distances from the spindle equator. These comparisons indicated that interactions of several factors, and not the motor axon alone, determine the form of motor endings. Intrafusal muscle fiber type is dominant to the motor axon in regulation of the number and depth of postsynaptic folds. Separation of the influence of the motor axon from the muscle fiber was less clear with respect to the size of ending. Complete expression of the muscle fiber-motor axon interaction reflected by the form of motor endings is dependent upon location of the ending relative to the sensory region. Both depth of the primary synaptic cleft and size of the soleplate of motor endings increased with increasing distance of the ending from the spindle equator. A system of classification of cat intrafusal motor endings that reflects the multiplicity of factors that determine the form of endings, and one that simplifies the current terminology, is proposed.     

Sensory activity affects sensory axon development in C. elegans

The simple nervous system of the nematode C. elegans consists of 302 neurons with highly reproducible morphologies, suggesting a hard-wired program of axon guidance. Surprisingly, we show here that sensory activity shapes sensory axon morphology in C. elegans. A class of mutants with deformed sensory cilia at their dendrite endings have extra axon branches, suggesting that sensory deprivation disrupts axon outgrowth. Mutations that alter calcium channels or membrane potential cause similar defects. Cell-specific perturbations of sensory activity can cause cell-autonomous changes in axon morphology. Although the sensory axons initially reach their targets in the embryo, the mutations that alter sensory activity cause extra axon growth late in development. Thus, perturbations of activity affect the maintenance of sensory axon morphology after an initial pattern of innervation is established. This system provides a genetically tractable model for identifying molecular mechanisms linking neuronal activity to nervous system structure.     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.     

Morphology and central synaptic connections of the efferent neurons innervating the crayfish hindgut

Summary The distribution, morphology and synaptic connections of the hindgut efferent neurons in the last (sixth) abdominal ganglion of the crayfish, Orconectes limosus, have been investigated using light and electron microscopy in conjunction with retrograde cobalt/nickel and HRP labeling through the intestinal nerve. The hindgut efferent neurons occur singly and in clusters, and are unipolar. Their axonal projections are uniform and consist of a thick primary neurite with typical lateral projections and limited arborization of varicose fibers in the ganglionic neuropil. They also send lower order axon processes to the ganglionic neural sheath, where they arborize profusely, forming a network of varicose fibers. The majority of the efferent neurons project to the anterior part of the hindgut. HRP-labeled axon profiles are found in both pre- and postsynaptic position in the neuropil of the ganglion. HRP-labeled axon profiles also establish pre- and postsynaptic contacts in the intestinal nerve root. All hindgut efferent terminals contain similar synaptic vesicle populations: ovoid agranular vesicles (50–60 nm) and a few large granular vesicles (100–200 nm). It is suggested that the hindgut efferent neurons in the last abdominal ganglion are involved in: (1) innervation of the hindgut; (2) central integrative processes; (3) en route synaptic modification of efferent and afferent signals in the intestinal nerve; (4) neurohumoral modulation of peripheral physiological processes.Fellow of the Alexander von Humboldt Stiftung     

Axo-axonal synapses in the frog tectum opticum

A quantitative electron-microscopic investigation of synaptic endings in large sections showed that about 50% of all axo-axonal synapses are located in the outer zone of the neuropil (layer 9) of the tectum opticum ofRana temporaria L. These synapses are more numerous in the rostral part of the tectum than the caudal. Hardly any axo-axonal synapses lie deeper than 50–60 µ Most axo-axonal synapses are located on axon endings of retinal ganglionic cells, for after degeneration of the optic nerve the number of these synapses is reduced by two-thirds. During ontogenetic differentiation and regeneration of the optic nerve axo-axonal synapses develop before axo-dendritic and their presynaptic processes have the normal structure and differ sharply from the bulbs of growth of the optic fibers. On this basis the central origin of most presynaptic processes forming these synapses is postulated. The results point to the possibility of presynaptic control over the effectiveness of action of the efferent axons, primarily optic, terminating in the outer zone of the frog tectum opticum.     

The Patterning of Neuronal Connections

SYNOPSIS. The final level of development in the vertebrate nervoussystem concerns the patterning, or modelling, of the connectionsof the postmitotic neurons at target tissues. The growth oftarget tissue and the sprouting of newly-arrived axons is followedby a regulated distribution of the sprouts, and often the developmentof specific contacts (synapses). Usually there are more sproutsthan can be accommodated; the result is competition for targetsites and regression of unsuccessful endings, leading to thecharacteristic pattern of connections. Studies particularlyof the reinnervation of denervated skin in salamanders and ratshave revealed a number of conditions and processes that influencecompetition. Spatial and temporal constraints restrict the sproutingof certain nerves but not others. Substances conveyed to nerveterminals by fast axoplasmic transport can "neutralize" sproutinginfluences believed to derive from target tissues, and can "mask"target features that nerves recognize; other substances areneeded for sprouting to occur at all. Increased impulse activityin certain nerves accelerates their sprouting. Intriguingly,regenerating nerves will recapture targets from sprouted endings.Most of the identified conditions and processes occur in theadult animal; they could therefore provide a basis for remodellingof neuronal connections. Remodelling occurs when the characteristicearly hyperinnervation of target tissues converts after birthto the adult pattern; in cutaneous "touch domes" targets areeliminated in addition to endings. Perhaps remodelling playsa role in the development of behaviour even in mature animals.     

Immunogold electron microscopic demonstration of glutamate and GABA in normal and deafferented cerebellar cortex: correlation between transmitter content and synaptic vesicle size

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.