Size-related differences in the branching pattern of the motor nerve terminals in triangularis sterni muscle of the mouse

A light microscopy morphometric study was performed in singly innervated synaptic areas of the triangularis sterni muscle of the normal adult Swiss mouse. Investigating mechanisms of the motor nerve growth control, we tested the hypothesis that significant differences in the nerve terminal branching pattern can be detected between different populations of nerve endings classified according to their arborization complexity or size. The main observations of this morphometric study are first, that the mean segment length of the terminal arborization between branch points behaves as an independent variable from the remaining parameters; the mean value of this parameter did not change in nerve endings of differing size and complexity. Secondly, the increase in size of the nerve endings is accompanied by a significant reduction in the mean length of the distal free-end segments. Results are discussed in the context of the possible regulatory mechanisms governing nerve terminal growth and remodelling.     

Submicroscopic Changes of the Nerve Endings in the Adrenal Medulla after Stimulation of the Splanchnic Nerve

The nerve endings of the adrenal medulla of the rabbit were studied under the electron microscope in the normal condition and after prolonged electrical stimulation of the splanchnic nerve. With a stimulus of 100 pulses per second for 10 minutes, there is an increase in the number of synaptic vesicles in the nerve ending. The mean number is of 82.6 vesicles per square micron in the normal and of 132.7 per square micron in the stimulated glands. With a stimulus of 400 pulses per second for 10 minutes, there is a considerable depletion of synaptic vesicles and other changes occur in the nerve endings. The mean number of vesicles is of 29.2 per square micron. These results are interpreted as indicative of an increased activity of the ending in one case, and as a diminished activity and fatigue of the synaptic junction in the other.     

Ultrastructure of the joint receptors in the frog (Rana temporaria).

The ultrastructure of sensory nerve endings was examined in joint capsules of large limb joints in three adult frogs (Rana temporaria). The joint receptors are represented by the only one kind of sensory nerve endings--by free nerve endings. The unmyelinized preterminal desintegrates into single terminals. This branching is bound on the most peripheral cell of the Schwann cell by means of mesaxons, they pass from the pericaryum of the Schwann cell peripherally. The branches of the nerve terminal are surrounded by a cover of 1...3 cytoplasmatic processes of the Schwann cell. The surface lamella is covered by a distinct basal membrane. Bundles of collagenous fibrils pass along the branches of the nerve terminal. Quite naked nerve endings were not observed. The axoplasma of the nerve terminal contains strikingly few cell organels. Besides axially passing neurofilaments and neurotubules only sporadic mitochondria and clear vesicles were observed. The accumulation of mitochondria, characteristic for the axoplasma of nerve terminals, was observed in no case. Free nerve endings which were found in the joint capsules of the frog belong among so called "free penicillate nerve endings".     

Cytosolic sialidase from the nerve endings of developing rat forebrain

The developmental profile of cytosolic sialidase from the nerve ending and the nerve ending-free compartments of rat forebrain was studied from birth to 150 days. Soluble extracts containing the cytosol from nerve endings and nerve ending-free tissue were separately prepared; the recovery of cytosol in the two extracts was followed by assaying lactate-dehydrogenase. In both cytosolic extracts the content of sialidase and lactate-dehydrogenase had a marked and progressive enhancement from the 5th to the 20th day of life and then maintained a constant level through adult life. However the rate of lactate-dehydrogenase and sialidase increase in the two cytosolic compartments was different. Lactate-dehydrogenase increased at a very similar rate in the two cytosols. Instead the rate of sialidase increase was greater in the cytosol from nerve endings from the 5th to the 10th day of life, and, inversely, in the cytosol from nerve ending-free tissue from 10 to 20 days. Between the 5th and 10th day of life the nerve ending cytosol underwent an enrichment of sialidase which was several times higher than that of lactate-dehydrogenase; in the other periods of forebrain development sialidase and lactatedehydrogenase moved in parallel.The variations with age of cytosolic sialidase from the nerve ending-free tissue seemed to follow the overall process of brain development. The nerve ending soluble sialidase would more specifically reflect formation of synaptic junctions in some brain regions in a well defined period of brain maturation.     

GABA and glutamate fluxes in developing nerve endings

Radiolabeled GABA and glutamate transport into 7 day, 14 day and adult cortical nerve ending preparations was examined. Transport was measured at several Na⁺ concentrations, 19, 27, 43 and 121 mM, and at two temperatures, 15 and 30°C. K_m and V_max values were calculated for all experimental conditions by means of Wilkinson (1961) analysis. A comparison of the day 14 and adult data shows higher K_m values at all Na⁺ concentrations on day 14 for both GABA and glutamate transport. In addition, the temperature dependence of transport was attenuated in the day 14 preparation. Finally, the specificity of GABA transport, as measured by the use of the transport inhibitors β-alanine and 2,4-diaminobutyric acid, was not different between the day 14 and adult preparations. Overall, it is concluded that both GABA and glutamate transport into day 14 nerve endings behave as if “adult” transporter molecules were existing in a more fluid lipid environment, which is the situation found in synaptic membranes prepared from day 14 nerve endings (Hitzemann and Johnson, 1983).Glutamate and GABA transport into 7 day nerve endings is complex and shows marked differences from the day 14 and adult data. Day 7 GABA transport was significantly more sensitive to β-alanine inhibition. Day 7 transport was more sensitive to Na⁺ manipulation and the temperature dependent kinetics show unique Na⁺ effects not seen in the day 14 or adult preparations. For example, at 19 mM Na⁺, 7 day glutamate transport was more temperature dependent than adult transport but as the Na⁺ concentration was increased the reverse was true. The opposite situation for temperature-Na⁺ effects was seen for GABA transport. Finally, no Ca⁺²-dependent component of GABA release could be found in 7 day nerve endings while a significant component was found at day 14. Overall, it is concluded that both glutamate and GABA fluxes in 7 day nerve endings differ both qualitatively from that seen in both day 14 and adult nerve endings.     

Morphometric study of the neuromuscular synapses in the adult rat with special reference to the remodelling concept

There is increasing morphologic evidence that neuromuscular synapses are not rigid structures in the mature muscles of adult animals. On the contrary, they may be submitted to a continuous process of remodelling. In silver-impregnated sternocleidomastoid muscles of the young adult rat, we measured synaptic parameters such as nerve terminal length, the number of branching points of terminal arborization, and muscle fiber diameter, and used a morphometric approach to explore specific questions concerning neuromuscular remodelling. Quantitative data indicate that: (a) The complexity and maturation of the nerve endings in this muscle are very variable and the increase in branching points is not paralleled by an increase in terminal length; (b) Muscle fiber diameter is related only marginally to presynaptic parameters; (c) Accessory ending formation occurs when the original ending does not reach the mean size of endings in singly innervated areas; (d) The complexity of individual endings at dually innervated junctions is smaller than the mean development of singly innervated synapses, indicating the existence of some mutual inhibitory influence between closely spaced endings. Morphometric results suggest a continuous process of synaptic formation in this adult muscle.     

Calretinin-immunoreactive laminar nerve endings in the laryngeal mucosa of the rat

The distribution of laminar nerve endings that contained immunoreactive calretinin was examined in the laryngeal mucosa of the adult rat. In whole-mount preparations, the immunoreactive laminar endings were distributed in the supraglottic region but not in the subglottic region. The laminar endings that arose from thick nerve fibers with or without swellings were identified as corpuscles with many variform terminal arborizations. They appeared to be located at the interface between the epithelium and the subepithelial connective tissue. The terminals were scattered under the basal lamina of the epithelium, and some of them were located within the epithelial layer. Immunoelectron microscopy revealed that both sub- and intraepithelial immunoreactive terminals that were filled with mitochondria were partly or totally ensheathed by Schwann cell processes. The denervation experiments, in which the superior laryngeal nerve was cut unilaterally or bilaterally, suggested that the laminar endings originate from the superior laryngeal nerve with strict ipsilateral innervation. The laminar endings might be associated with detection of changes in pressure in the laryngeal cavity or chemical stimuli.     

Immunohistochemical demonstration of calbindin-containing nerve endings in the rat esophagus

Immunoreactivity for calbindin was found in nerve endings with irregular laminar shapes in the rat esophagus. In the myenteric ganglia, laminar endings of a range of sizes formed a complex network and appeared to lie at the surface of the ganglion. The myenteric ganglia that contained nerve endings were most abundant in the upper portion of the eosphagus, their number decreasing orally to anally. Calbindin-immunoreactive nerve cell bodies were scattered throughout the esophagus. Laminar terminals were found in the connective tissue of the lamina propria immediately beneath the epithelium and in the muscularis mucosae. Occasional nerve branches formed a network of aborizing endings that surrounded part of the submucosal arterioles. Immunoreactive nerve endings in the mucosa and submucosa were present only in the upper part of the cervical esophagus. Unilateral vagotomy caused a remarkable decrease in the number of the myenteric ganglia containing the calbindin-immunoreactive laminar endings after 15 days or survival; in some of ganglia, the laminar structures disappeared and nerve endings showing weak immunoreactivity had an indistinct appearance, so that the outline of the ganglia became obscure. In operated rats at 24 days, the number of innervated ganglia was about half that in normal rats. However, there was no change in the morphology and the occurrence of the immunoreactive laminar structures in the mucosa and submucosa after denervation. The results show that many of the laminar endings that are immunoreactive for calbindin in the myenteric ganglia are derived from the vagus nerve. Thus, the calbindin-immunoreactive nerve endings with laminar expansions that are found in the rat eosphageal wall could be sensory receptors.     

Pattern of arborization of the motor nerve terminals in the fast and slow mammalian muscles.

A silver impregnation method and a morphometric approach were used to define differences existing in the motor nerve terminal branching pattern between a fast-twitch muscle (extensor digitorum longus) and a slow-twitch one (soleus) of the normal adult rat. Because no single measure can describe precisely all geometrical properties (ie both topology and metrics) of the nerve terminals, we evaluated morphologic parameters defining length and angular characteristics in the different terminal segments classified according to their centrifugal order. The main results indicate that the distal free-end segments in the extensor digitorum longus muscle are shorter and less divergent than in the soleus nerve terminals. The endings in the two muscles have different fractal dimensions. Findings are discussed in the context of the hypothetical mechanisms governing motor nerve terminal size and complexity.     

Regulation of acetylcholine synthesis in presynaptic endings of cholinergic CNS neurons

Data on acetylcholine (ACh) synthesis in nerve cells are summarized and the mechanism of regulation of this process is described. Under conditions of relative rest on moderate synaptic activity the ACh concentration in the compartment of its synthesis in cholinergic nerve endings is probably maintained at a level corresponding to equilibrium of the reaction catalyzed by the enzyme choline-acetyltransferase (CAT). ACh release is followed by its transport from the compartment of synthesis into the compartment of secretion and automatic resynthesis of new ACh, until equilibrium is restored in the compartment of synthesis. At the same time synaptic activity and ACh release promote synthesis of new ACh by the following pathways. First, a fall in the ACh concentration in the nerve endings disinhibits carriers for choline, and facilitates choline transfer from the extracellular fluid into the cell in accordance with the electrochemical gradient. Second, hydrolysis of liberated ACh increases the choline concentration in the extracellular fluid in the neighborhood of the nerve endings. Third, postactivation hyperpolarization of the nerve endings facilitates transport of choline and an increase in its concentration in the nerve endings. Fourth, there are grounds for considering that stimulation of muscarine receptors promotes a further increase in the choline concentration in the region of the nerve endings by intensification of phosphatidylcholine hydrolysis in postsynaptic cells. Fifth, a decrease in the acetyl-CoA content on account of ACh resynthesis increases pyruvate dehydrogenase activity and acetyl-CoA production. Sixth, it is possible that an increase in the Ca⁺⁺ concentration in nerve endings promotes direct transport of acetyl-CoA from the mitochondria into the cytosol of nerve endings, where ACh is synthesized. It is postulated that under conditions of intensive synaptic activity the rate of supply of acetyl-CoA and choline and also CAT activity in the nerve endings may be factors limiting the velocity of ACh resynthesis.Institute of Physiology, Czechoslovak Academy of Sciences, Prague. Translated from Neirofiziologiya, Vol. 16, No. 5, pp. 603–611, September–October, 1984.     

Shared and unique roles of CAP23 and GAP43 in actin regulation, neurite outgrowth, and anatomical plasticity

CAP23 is a major cortical cytoskeleton-associated and calmodulin binding protein that is widely and abundantly expressed during development, maintained in selected brain structures in the adult, and reinduced during nerve regeneration. Overexpression of CAP23 in adult neurons of transgenic mice promotes nerve sprouting, but the role of this protein in process outgrowth was not clear. Here, we show that CAP23 is functionally related to GAP43, and plays a critical role to regulate nerve sprouting and the actin cytoskeleton. Knockout mice lacking CAP23 exhibited a pronounced and complex phenotype, including a defect to produce stimulus-induced nerve sprouting at the adult neuromuscular junction. This sprouting deficit was rescued by transgenic overexpression of either CAP23 or GAP43 in adult motoneurons. Knockin mice expressing GAP43 instead of CAP23 were essentially normal, indicating that, although these proteins do not share homologous sequences, GAP43 can functionally substitute for CAP23 in vivo. Cultured sensory neurons lacking CAP23 exhibited striking alterations in neurite outgrowth that were phenocopied by low doses of cytochalasin D. A detailed analysis of such cultures revealed common and unique functions of CAP23 and GAP43 on the actin cytoskeleton and neurite outgrowth. The results provide compelling experimental evidence for the notion that CAP23 and GAP43 are functionally related intrinsic determinants of anatomical plasticity, and suggest that these proteins function by locally promoting subplasmalemmal actin cytoskeleton accumulation.     

Morphometric analysis of hypoxia-induced synaptic activity in intrapulmonary neuroepithelial bodies

Summary A morphometric analysis has demonstrated ultrastructural changes induced by hypoxia in the epithelial cells and the intracorpuscular nerve endings of the presumed chemoreceptive intrapulmonary neuroepithelial bodies (NEB) of neonatal rabbits.Acute hypoxia stimulates an exocytosis of epithelial dense-core vesicles (DCV) at the level of the morphologically afferent or sensory (type 1 a) intracorpuscular nerve endings of the NEB. Assuming the epithelial cells to be chemoreceptive, this phenomenon could represent a transduction of sensory stimuli.In the morphologically efferent or motor (type 2 and type 1 b) intracorpuscular nerve endings of the NEB, acute hypoxia causes a depletion of synaptic vesicles and an increase in the amount of membrane-bounded cisternae and multivesicular bodies, suggestive of an enhanced synaptic activity of these nerve endings. It is proposed that the chemoreceptor cells could thus in turn be modulated centrifugally by their efferent-like intracorpuscular nerve endings.It has been proposed in our earlier studies that the NEB probably are intrapulmonary chemoreceptors with local secretory activities, reacting to the composition of the inhaled air. By the release of serotonin and peptide substances they may produce a local vasoconstriction in hypoxically aerated lung areas, enabling an intrapulmonary regulation of the V/Q ratio. The present study provides evidence that, in addition to this local effect, NEB could generate centripetal nerve impulses via exocytosis of epithelial DCV at the afferent-like intracorpuscular nerve endings. At the same time they could be modulated by the CNS via their efferent-like intracorpuscular nerve endings.With respect to their innervation, numerous similarities appear to exist morphologically and functionally between the carotid body and the intrapulmonary NEB.     

Correlative ontogenetic development of catecholamine- and LHRH-containing nerve endings in the median eminence of the rat

Summary The ontogenetic development of catecholamine (CA)-and LHRH-containing nerve endings in the median eminence of the rat was investigated by combining fluorescence histochemistry and immunohistochemistry in the same tissue section. LHRH-terminals appeared earlier than CA-terminals and were already detectable in the lateral part of the external layer of the central ME on the first day after birth. CA-nerve endings were first seen in a corresponding region of the ME on the seventh postnatal day. At this stage both types of terminals showed the earliest manifestation of a correlative pattern of their distribution. Subsequently the development of both types of nerve endings proceeded rapidly, and at 14 days their distribution pattern corresponded to that in adult animals. The authors conclude that at this stage the CA-neurons play a constant and significant role in the release of LHRH into the portal capillaries. The correlation between both types of nerve endings and the ontogenetic development of the capillary plexuses of the hypophysial portal system is discussed.This work was supported in part by a grant (No. 248093, 321426) from the Ministry of Education, Science and Culture, Japan     

Cocaine and lithium: neurobiological antagonism in the serotonin biosynthetic system in rat brain.

Cocaine reduced the uptake and conversion of tryptophan to serotonin in rat brain striate tissue and enhanced tryptophan hydroxylase activity in lateral midbrain cell bodies and striate nerve endings. Lithium augments the uptake and conversion measures and reduces the enzyme activity in cell bodies and nerve endings. The cocaine effects on all four measures were antagonized by three days of lithium pretreatment.     

Combination of non-specific cholinesterase histochemistry and immunofluorescence staining for the study of the sensory innervation of skin and muscle

Summary In the present study we describe the application of the non-specific cholinesterase (nChE) histochemical method for the detection of encapsulated sensory nerve endings prior to immunofluorescence staining of the sensory nerve fibres. The nChE staining of Schwann-derived structures surrounding sensory terminals allowed us to identify unequivocally the sensory corpuscles in the skin and the muscle proprioceptors (muscle spindles and Golgi tendon organs) in longitudinal sections of muscle tissue. The nChE staining of sensory nerve endings and immunofluorescence-labelled nerve fibres and their terminals could be viewed and photographed in the same section using appropriate filters. Since nChE activity persists in terminal Schwann cells for a long time after loss of the sensory axons, this combined enzyme- and immunohistochemical approach is also useful for experimental studies involving denervation and re-innervation of sensory nerve endings.     

Quantitative evaluation of sensory nerve endings in hypertrophy of labia minora pudendi in women.

The authors studied the types of sensory nerve endings in hypertrophic labia minora pudendi in women aged between 18 and 69. The nervous tissue was impregnated with silver nitrate by Lavrentyev's modification of the Bielschowski-Gross method. The sensory nerve endings were divided into the following groups: free endings and arborizations, spray-like endings, seven types of clew-like nerve endings, and Pacinian corpuscles. It is the clew-like endings that absolutely predominate, they were 2,027 in number. The arborizations and spray-like endings found were 105 in number and only 4 of Pacinian corpuscles were found. As compared with normal labia there are no principal differences among the types of sensory nerve endings. The main difference consists in the rate of occurrence (89.6 as against 57.8) and in the thickness of the capsule, while the nerve clew proper does not grow in diameter. Therefore, we are of theopinion that a more frequent occurrence and larger size of the capsule is associated with local changes in the organ (especially with hypertrophy of the connective tissue). We observed no differences due to age either in the occurrence of the individual types or in the thickness of the capsule. Great individual variability in both directions was seen. The findings corroborated again the rare occurrence of meissner's endings in the labia minora in woman. Therefore it may be assumed that it is rather a random finding and that it is a type of clew-like nerve ending. Finally, we presume that the individually varying rate of occurrence of the nerve endings in the external genitals could also affect general sensitivity in this region, including sexual sensitivity.     

The ultrastructure of the endings of nerve fibers of ordinary hairs of mammals under normal conditions and during degeneration and regeneration

At ultrastructural level, three zones are distinguished of the palisade endings of nerve fibers, most of them in contact with the basement membrane of the outer epithelial sheath of the radix pili on rat muzzle. After division of the supplying nerve (n. infraorbitalis) in test animals of the same species, its nerve fibers and palisade endings undergo degeneration, followed by regeneration within 4-5 months. Changes in the ultrastructure of the mentioned nerve elements are thoroughly studied and demonstrated in the course of the two processes. While within 6...12 days of the operation, the palisade endings degenerate, 130 days after the intervention most of them are restored and exhibit an appreciable difference relative to their typical ultrastructure in normal state. The Schwann's cells processes, enveloping degenerated and regenerated palisade endings, similarly display marked fine-structure alterations.     

Further studies of the transport of protein to nerve endings

Mice were injected intracerebrally with [l-¹⁴C]leucine, and the specific activities of subcellular fractions of brain and effractions of isolated nerve endings were determined. There was a progressive increase in the specific activity of protein associated with isolated nerve endings after incorporation of [l-¹⁴C]leucine into whole brain protein had terminated. Although, the incorporation of [¹⁴C]leucine into soluble protein of whole brain did not differ significantly in mice which were 3 months or 1-year old, the subsequent increase in specific activity of soluble protein isolated from nerve endings was significantly greater in the younger animals; 6-month-old mice were intermediate. Therefore, changes in some aspect of the transport of protein to nerve endings is altered even after sexual maturity. Anaesthetization with pentobarbitone during incorporation of [¹⁴C]leucine into protein, and inhibition of protein synthesis with acetoxycycloheximide after incorporation of [¹⁴C]leucine was complete, did not interfere with the subsequent appearance of radioactive protein at the nerve ending. Evidence is presented for the transport, from a proximal site of synthesis, of protein associated with particulate components of the nerve ending, including synaptic vesicles.     

RESOLUTION OF THREE DISTINCT POPULATIONS OF NERVE ENDINGS FROM RAT BRAIN HOMOGENATES BY ZONAL ISOPYCNIC CENTRIFUGATION

Conditions have been established for the fractionation of subcellular components of rat forebrain homogenates by zonal isopycnic equilibration in continuous sucrose density gradients using a B-XIV rotor. The fractions were analyzed biochemically and by ultra-structural morphometry. Starting from postnuclear supernates of forebrain homogenates, it has been possible to resolve three distinct populations of nerve endings from one another, as well as from free mitochondria and myelin fragments. The three types of nerve endings differ in their apparent specific gravity, their biochemical properties, and their ability selectively to accumulate exogenous transmitter substances in vitro. These three particle populations are likely to represent, in order of increasing modal equilibrium density, (a) cholinergic nerve endings, characterized by their high content of acetylcholine, (b) γ-amino butyric acid (GABA)-containing nerve endings with high glutamate decarboxylase activity and the ability to accumulate exogenous GABA, (c) adrenergic nerve endings that accumulate exogenous dopamine and noradrenaline and exhibit high monoamine oxidase activity.     

Ultrastructure of the synaptic endings in nerve tissue transplants developing in the anterior chamber

Embryonic septum of hippocampus was grafted into the anterior eye chamber (AEC) of adult recipient rats. The fine structure and distribution of synaptic endings were studied in the hippocampus (HC) and septum (ST) grafts developing in oculo for 3-4 months. On the basis of the structure of postsynaptic regions, asymmetrical and symmetrical synapses are distinguished, whose distribution on the body and dendrites of hippocampal and septal neurons is basically similar with that in situ. As in vivo, axo-somatic, axo-dendrite and axo-spine forms of synaptic endings have been observed. Neuropile has, basically, normal structure, judging by the ratio of nerve and glial elements, but sometimes dendro-dendrite contacts and glomerular-like synaptic structures are observed which are not characteristic of the studied brain regions. Besides, the grafts contain an increased number of serial and tangential synapses, as well as axonal terminals with the signs of growth cones. The observed structural deviations appear to be due to incomplete tissue maturation in the absence of normal afferentation.