Ultrastructure of substance P-immunoreactive terminals and their relation to vascular smooth muscle cells of rat small mesenteric arteries.

Mesenteric arteries of the rat are surrounded by a plexus of primary afferent nerve terminals which contain both substance P (SP) and calcitonin gene-related peptide (CGRP). The ultrastructural arrangement of the innervation was studied in second-order branches of the rat mesenteric artery using immunohistochemical labelling with antibodies against SP. The structure and distribution of SP-immunoreactive (SP+) and SP-negative (SP-, i.e., virtually all noradrenergic) axons and their terminals within the adventitia of the artery have been determined. Sixteen percent of axons and 22% of varicosities in the perivascular plexus were SP+. Most of the SP+ varicosities lay between 0.4 and 2 microm from the smooth muscle cells, whereas most SP- varicosities lay much closer to the vessel (i.e., <1 microm). SP+ varicosities typically contained the same number and size of small synaptic vesicles and mitochondria as SP- varicosities, but there were more large dense-cored vesicles in the SP+ varicosities. Unlike SP- varicosities, the peptidergic varicosities did not show clustering of synaptic vesicles toward one part of the axon membrane, and none of them formed junctions with the smooth muscle cells. Close relationships between SP+ and SP- varicosities lacked any detectable structural specialization. The arrangement of SP+ (primary afferent) terminals and their association with vascular smooth muscle cells indicates that peptide released from afferent terminals must diffuse further than noradrenaline from sympathetic terminals to reach the vascular smooth muscle.

Proportions and structure of contacting and non-contacting varicosities in the perivascular plexus of the rat tail artery.

Most sympathetic postganglionic noradrenergic varicosities of the perivascular plexus of small muscular arteries in laboratory mammals make contact with the outer smooth muscle cells of the media at neuromuscular junctions. These neurovascular junctions have most of the characteristics of those in skeletal muscle. In the rat tail artery, which bears a particularly dense perivascular plexus, many studies indicate that both purinergic and noradrenergic mechanisms underlie neurally mediated vasoconstriction. We have examined the relationship of large axonal varicosities to the smooth muscle surface of proximal parts of this vessel using three-dimensional reconstructions from serial thin sections photographed in the electron microscope. Unlike in small arterioles, less than 50% of the large photographed in the electron microscope. Unlike in small arterioles, less than 50% of the large varicosities lying within 1 micron of the outer surface of this artery were found to make neuromuscular junctions. In some non-contacting varicosities, accumulations of synaptic vesicles were aggregated toward axonal membrane which was bare of Schwann cell toward the vessel surface. Prejunctional membrane specializations were detected at 20% of contacting and 12% of non-contacting varicosities. All of the latter lay close (< 350nm) to the smooth muscle. These anatomical data suggest that, in the rat tail artery, transmitter release by exocytosis may occur from both types of varicosity.

Ultrastructural analysis of sympathetic neuromuscular junctions on mesenteric veins of the guinea pig.

This study reports on the detailed ultrastructure of sympathetic postganglionic varicose axon terminals on mesenteric veins leading from the ileum of the guinea pig and in particular the structural arrangement of the varicosities with venous smooth muscle cells. The response to nerve stimulation in veins has a long time course and it has been suggested that this reflects a wide separation between the site of transmitter release and the receptors on the effector cell membrane. The aim of this study was to determine the distance between individual sympathetic varicosities and smooth muscle cells in mesenteric veins. Fluorescent histochemical preparations of the sympathetic innervation of the different branches of mesenteric veins indicate the branching network of varicose axons around the vessel to be relatively dense. Electron micrographs show the innervation to be confined to the adventitia close to the medio-adventitial border and to be predominantly catecholaminergic. A serial section ultrastructural analysis of the relationship of the varicosities with the outer smooth muscle cells showed that almost all (98%) of the exposed axon varicosities in the adventitia formed neuromuscular junctions. Three-dimensional reconstructions from serial sections of individual varicosities have shown that the junctions have structural specialisations identical to neuromuscular junctions described on arterial vessels and similar to those found at skeletal neuromuscular junctions. The density of neuromuscular junctions on the veins was found to be similar to that on the corresponding artery in the same animal. We suggest that in veins, noradrenaline is released focally at neuromuscular junctions.

An ultrastructural analysis of the sympathetic neuromuscular junctions on arterioles of the submucosa of the guinea pig ileum.

The relationship of the varicosities of sympathetic postganglionic nerve terminals to the smooth muscle cells of arterioles in the submucosa of the guinea pig ileum has been investigated quantitatively by electron microscopy. Longitudinal sections were cut through arterioles about 50 micron in diameter after fixation in vitro or in situ under pressure. About 13% of the varicosities in individual ultrathin sections made contact with the outer surface of the smooth muscle cells. The neuromuscular junctions resembled those in skeletal muscle: the basal laminae of the axon bundle and of the smooth muscle were fused, and synaptic vesicles were accumulated close to the region of fusion. When individual varicosities were examined in serial sections, 92% and 83% in two preparations were found to form junctions of this kind. Most of the noncontacting varicosities were bare of Schwann cell toward the arteriolar surface and separated from it by less than 200 nm. Almost all axon profiles contained synaptic vesicles with electron dense cores after exposure to 5-hydroxydopamine. In electrophysiological experiments, ionophoretic application of noradrenaline to the arteriolar surface along the nerve bundles (demonstrated subsequently by fluorescence histochemistry) produced responses resembling those evoked by nerve stimulation. These anatomical and physiological data, taken together with the evidence for quantal release in this preparation (see Hirst et al., '85), suggest that neuromuscular transmission involves the rare release of a quantum of noradrenaline at discrete points on the smooth muscle membrane.

Sympathetic innervation of renal and extra-renal arterial vessels.

The innervation of arterial vessels has been analyzed using serial thin sections examined in the electron microscope. Sympathetic postganglionic noradrenergic axons have been found to make neuromuscular junctions on the outermost cells of the media of virtually all muscular arterial vessels with diameters between 20 microns and 1 mm in rat, rabbit and guinea pig. Junctions are formed by the majority of varicosities which are not covered by Schwann cell on the side towards the artery surface. The junctions resemble those on skeletal muscle fibers, having a single layer of basal lamina in the synaptic cleft. Although post-junctional specializations have not been identified, 15 to 20% of junctions show electron dense regions of the prejunctional membrane with associated clumps of synaptic vesicles. The form of the varicose axons differs, and three distinct types have been distinguished on morphological grounds. Two of these occur in the juxtaglomerular region of the kidney, with characteristic distributions of junctions which differ between the afferent and efferent arterioles. The presence of numerous close junctions has revolutionized our thinking about how sympathetic neuroeffector transmission occurs in arterial vessels.

Electrical coupling between smooth muscle and endothelium in arterioles of the guinea-pig small intestine.

Equations describing the steady-state passive electrical properties of arterioles have been derived. The arteriole was modelled as having two thin layers of cells (muscle and endothelium) with strong electrical coupling between cells within a layer and variable coupling between the layers. The model indicated that spread of membrane potential changes was highly dependent on the thickness of cells within the layers. The model was also used to identify the optimal experimental strategy for detecting coupling between the two layers, and experiments were carried out on arterioles from the guinea-pig small intestine. Thickness of the endothelial layer was measured using electron microscopy and was found to be around 0.5 microm. Electrical input resistance was measured in intact arterioles and compared to input resistance of arterioles from which the endothelium had been removed. The experiments confirmed that there was a strong electrical coupling between the muscle and endothelium in these vessels.

Ultrastructure of sympathetic axons and their structural relationship with vascular smooth muscle.

This review focuses on the more recent findings of the structure of sympathetic postganglionic axons and the association of their varicose terminals with vascular smooth muscle. These studies have investigated the innervation of a wide range of vessels from different regions of the vasculature in the rat, guinea pig and rabbit and have predominantly used serial sections and computerised three-dimensional reconstructions of entire varicosities. They have shown, contrary to previous studies conducted in the 1960s and 1970s, that sympathetic axon varicosities commonly form structurally specialised neuromuscular junctions with vascular smooth muscle cells of most resistance arteries and some small veins. In addition, they have shown that most axon varicosities innervating small arterioles and small mesenteric veins form neuromuscular junctions, indicating that neurotransmitter is primarily released at such neuromuscular junctions. This review discusses the structure of sympathetic neuromuscular junctions, their development, structural diversity and distribution on vessels from different regions of the vasculature. These more recent structural findings and their possible significance for our understanding of mechanisms involved in neural transmission in blood vessels is discussed.

Force--sarcomere-length relation and filament length in rat extensor digitorum muscle.

Relations between sarcomere length (SL) and force (F) were studied in ten fiber bundles (six to twenty fibers) from rat extensor digitorum muscles. A bundle (60 micron by 200-300 microns) was mounted in a glass covered perfusion chamber containing modified Krebs Henseleit buffer at 25 degrees C, oxygenated with 95% O2, 5% CO2 and pancuronium bromide (8 mg/1). F ( Disa 51E 01 transducer) and SL (laser diffraction and light microscopy) were measured; the latter could be controlled by a servomotor system. 200-500 ms tetanic stimulus trains were applied via platinum electrodes parallel to the muscle with 20% above maximal intensity, 160 Hz frequency and 1 ms duration of pulses. Tetani were at 2 min intervals. F attained a steady value 100 ms after the start of the tetanus at 2.0-2.5 microns SL and 350 ms at 3.5 microns SL. Active force, measured during tetani in which sarcomere length was held constant, was maximal between SL = 2.15 microns and 2.65 microns and declined in linear fashion with SL to zero at SL = 3.90 microns. Active force at SL = 2.00 microns was 95% of maximal force. Passive force was manifest above SL = 3.10 microns and was 10% of maximal force at 3.80 microns. Eight similar bundles were processed conventionally for electron microscopy (Philips EM 201A ) while SL was measured during the processing steps. Measurements were made from micrographs of longitudinal sections. SL measured from the micrographs were consistent with the observed shrinkage (5%). Actin periodicity was 41.5 +/- 0.19 nm; twenty-seven periods per actin filament were found. Filament lengths were corrected for an assumed actin periodicity of 39 nm. Actin length was 1.13 +/- 0.013 micron; myosin length was 1.53 +/- 0.015 micron. Bare zone was 0.17 micron +/- 0.01 micron. These filament lengths would give optimum overlap at SL between 2.26 and 2.43 microns and a linear decrease to zero with increasing SL from 2.43 microns to 3.79 microns. Actual force was consistently higher than predicted by overlap and force was maintained to both the left and the right of the predicted plateau.

Development of neuromuscular junctions on small mesenteric arteries of the rat.

This ultrastructural study has investigated the development of the innervation of second order mesenteric arteries from the ileum region of the rat intestine, particularly, the time course of the formation of the plexus of varicose axons around the arteries, and the formation of autonomic neuromuscular junctions. The time points studied were postnatal days-2, -4, -8 and -13. This study has revealed that the formation of neuromuscular junctions with mature structural characteristics occurred at;2 weeks postnatal. The plexus of varicose axons developed predominantly between day-4 and day-13, which agrees with previous light microscopy studies of catecholamine containing nerves around similar vessels. At day-2 and day-4, the axons lacked varicosities and were mainly contained in large bundles located in the outer region of the adventitia. The medio-adventitial border consisted of a dense layer of extracellular matrix and fibroblasts. By day-8, there were more axons and most were distributed in smaller bundles. Some had grown through the adventitia to lie at the medio-adventitial border and axon varicosities were also observed. Some varicosities had formed rudimentary neuromuscular contacts. By day-13, there were significantly more contacting varicosities compared to day-8. They were structurally more mature, being twice the size with three times the number of synaptic vesicles and consistently contained a mitochondrion. Conversely, the neuromuscular contact areas were similar at both time points. Some organisation of the synaptic vesicles associated with the prejunctional membrane, was evident in varicosities at day-8 but there were no presynaptic membrane specialisations similar to the putative neurotransmitter release sites found at mature skeletal neuromuscular junctions. The aggregation of small vesicles at the prejunctional membrane was more pronounced in neuromuscular junctions at day-13 with some having presynaptic membrane specialisations. Comparison of the structure of developing autonomic neuromuscular junctions with that of skeletal neuromuscular junctions has revealed a number of similarities.

Frequency of neuromuscular junctions on arteries of different dimensions in the rabbit, guinea pig and rat.

The medio-adventitial border of a variety of perfusion-fixed arteries of young adult rabbits, guinea pigs and rats has been studied in the electron microscope. The arterial media in the different vessels ranged from 2 to 25 cells thick. Neuromuscular junctions, defined as axon varicosities containing synaptic vesicles closely apposed to the outer surface of smooth muscle cells, with only a single layer of basal lamina intervening between axon and muscle membranes, were identified in all three species. Junctions were found in most vessels less than 1 mm in diameter with a frequency ranging from 8,000-150,000 junctions per square millimeter of smooth muscle surface, the number generally increasing with decreasing arterial diameter. These small arteries were mostly, but not exclusively, muscular rather than elastic. In large arteries, such as abdominal aortae and some elastic arteries lying close to the heart (e.g. the carotid), no junctions were found. In a few vessels, such as guinea pig basilar (muscular) and rat and guinea pig superior mesenteric (elastic) arteries, junctions were found infrequently (1,000-4,000/mm2). The data indicate that all muscular arteries in rats and guinea pigs, and most muscular arteries in rabbits, receive innervation in the form of sympathetic neuromuscular junctions. Whilst a few elastic vessels are sparsely innervated with junctions, some are surrounded by axon bundles containing vesicle-filled varicosities. The physiological significance of the latter is obscure.

The form of sympathetic postganglionic axons at clustered neuromuscular junctions near branch points of arterioles in the submucosa of the guinea pig ileum.

In fluorescence histochemical studies of the sympathetic innervation of the arterial vessels of the submucosa of the guinea pig ileum, we have identified clusters of varicosities overlying arteriolar branch points. These were particularly obvious on the small arterioles less than 45 microns in diameter, the non-branching regions of which generally lack much other innervation. Serial reconstruction from electron micrographs of axon bundles from the region of arteriolar branch points revealed the form of the varicose axons. Branching of axon bundles sometimes involved branching of individual axons. Within a cluster, most axons had several varicosities along lengths as short as 3 micron. The varicosities were very irregular in size and shape. Most large varicosities (greater than 1 micron in diameter) that were bare of Schwann cell covering (73%) formed neuromuscular junctions with basal lamina intervening between axon and muscle membranes. A smaller proportion (44%) of small varicosities (0.5-1.0 micron diameter) also formed junctions. Of all bare varicosities, 37% did not form contracts. In several of the large varicosities, prejunctional membrane specializations were identified over part of the neuromuscular junctions.

Extended angiotensin converting enzyme inhibition changes the innervation of renal glomerular afferent arterioles.

Chronic inhibition of the angiotensin I converting enzyme (ACE) with enalapril, results in a phenotypic change of the medial cells of renal afferent arterioles from contractile smooth muscle cells to renin containing epithelioid cells. In normal animals, the density of the innervation of the juxtaglomerular renin containing epithelioid cells is much lower compared to the contractile cells. The effector tissues are known to play an important role in determining the pattern and density of their innervation. In this study, we tested the hypothesis that the density of the innervation of the afferent arteriole smooth muscle cells decreases when they change their phenotype from contractile to renin containing epithelioid cells. The results show that the density of the innervation had significantly increased and the association of the terminals with the smooth muscle cells had changed. There were significantly more varicosities around renal afferent arterioles from rabbits treated with enalapril (10 microg/kg/h) for 6 weeks (mean +/- SEM = 634 +/- 175 x 10(3)/mm2 vessel surface, cf. 329 +/- 69 x 10(3)/mm2 vessel surface in untreated rabbits, P = 0.05), with the number of neuroeffector junctions remaining the same (124 +/- 14 and 164 +/- 32 x 10(3)/mm2 vessel surface) and significantly more non-contacting varicosities (i.e. lying > 100 nm from the medial cells) (74 +/- 5% and 25 +/- 7%, respectively; P = 0.003). Thus, there was no reduction in the innervation of afferent arterioles in which the smooth muscle cells had changed phenotype in response to enalapril treatment as hypothesised. Instead, it would appear that proliferation of the innervation had occurred, with the formation of additional varicosities but these varicosities failed to form neuromuscular junctions. This study has identified a form of neural plasticity in the kidney that has not previously been described.

Distribution of sympathetic neuroeffector junctions in the juxtaglomerular region of the rabbit kidney.

Two structurally distinct types of sympathetic axon (Type I and Type II) have recently been identified in the renal cortex of the rat and the rabbit. This study describes the distribution and density of the neuroeffector junctions made by these two types of axon on the different tissues from the juxtaglomerular region of the rabbit renal cortex. Immunohistochemical studies showed that tyrosine hydroxylase-positive axons were located only in regions adjacent to the arteries and arterioles in the renal cortex. Ultrastructural studies of the juxtaglomerular region indicated that both types of axon formed junctions on vascular smooth muscle cells, epithelial cells of proximal tubules and renin-secreting granular epithelioid cells. The density of neuromuscular junctions (18 x 10(3)/mm2 of vessel surface) was more than twice as high on the afferent arteriole as on the efferent arteriole or proximal tubules immediately adjacent to the glomerular arterioles (both about 6 x 10(3)/mm2). The junction density on granular epithelioid cells was much lower (about 2 x 10(3)/mm2) and were rarely observed on the distal tubule. Afferent arterioles preferentially received junctions from Type I axons at a relatively high density (14.2 x 10(3)/mm2) whereas junctions formed by Type II axons were less selectively distributed and occurred at lower densities on all other tissues (range, 1-6.3 x 10(3)/mm2). Presynaptic membrane specialisations were identified only at junctions on arterioles and granular epithelioid cells and occurred more frequently at Type I than at Type II junctions. The data suggest that the predominant effect of the sympathetic innervation in the juxtaglomerular region of the renal cortex is on the afferent arteriole and that the two axon types within the kidney may have different functions.

Two types of sympathetic axon innervating the juxtaglomerular arterioles of the rabbit and rat kidney differ structurally from those supplying other arteries.

Ultrastructural analyses of serial thin sections have revealed two structurally different types of sympathetic axon innervating the afferent and efferent juxtaglomerular arterioles and the intralobular arteries in the outer cortex of the rabbit kidney. Both types of axon have also been found in association with an afferent arteriole in rat kidney. One axon type consists of relatively large diameter unmyelinated axons bearing varicosities in the form of slight expansions. The varicosities have a distinct structural zonation: synaptic vesicles occupy the expansion which faces the smooth muscle cells, whereas the rest of the axon is filled with numerous microtubules. The other axon type has varicosities containing vesicles and mitochondria but few microtubules. The varicosities are generally small and the intervaricosities very thin. The relationship of both axon types with support cells and/or basal lamina is sometimes poorly defined. Both axon types are catecholaminergic as their vesicles take up 6-hydroxydopamine and both types form junctions with arteriolar smooth muscle cells. As well as differing from each other, both types of intrarenal axon differ in several respects from those which innervate other arterial vessels.