Susan E. Luff

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

SummaryUltrastructural 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.

Differential neural control of glomerular ultrafiltration

1. The renal nerves constrict the renal vasculature, causing decreases in renal blood flow (RBF) and glomerular filtration rate (GFR). Whether renal haemodynamics are influenced by changes in renal nerve activity within the physiological range is a matter of debate.

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.

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.

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.

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

SummaryIn 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 < 45 μm 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 μm. The varicosities were very irregular in size and shape. Most large varicosities (>1 μm 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 μm 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.

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 μm from the smooth muscle cells, whereas most SP− varicosities lay much closer to the vessel (i.e., <1 μm). 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. J. Comp. Neurol. 416:277–290, 2000.

Selective Increase in Renal Arcuate Innervation Density and Neurogenic Constriction in Chronic Angiotensin II-Infused Rats

Abstract—This study investigated the effects of angiotensin II “slow pressor” hypertension on structure and function of nerves supplying the renal vasculature. Low-dose angiotensin II (10 ng/kg per minute, initially sub-pressor) or saline vehicle was infused intravenously for 21 days in rats, and the effects were compared in renal and mesenteric arteries. Mean arterial pressure averaged 12±2 mm Hg higher than in vehicle-infused rats at 21 days. Using electron microscopy, the innervation density of renal arcuate, but not mesenteric arteries of equivalent size, was significantly higher in angiotensin II-infused than in vehicle-infused rats. Functional testing on a pressure myograph revealed that constrictions evoked by nerve stimulation in arcuate arteries were 2.3±0.7-fold greater in vessels from angiotensin II-infused compared with vehicle-infused rats (P <0.0001), whereas there was no significant difference in nerve-induced constrictions in mesenteric arteries. Sensitivity to and maximum amplitude of constrictions evoked by phenylephrine were not different in renal or mesenteric arteries between groups, suggesting that the increased neurally evoked constriction in renal arcuate arteries was not caused by postsynaptic changes. Endothelium-dependent vasorelaxation and the vessel wall physical properties were not different between the two groups in either artery. Thus, angiotensin II infusion appeared to evoke renal-specific increases in vessel innervation and increased vasoconstriction to nerve stimulation. These changes appear early and occur before changes in renal endothelial function are apparent. Thus, “slow pressor” angiotensin II hypertension is associated with increased renal innervation, compatible with a pathogenetic role.

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 catecholamne 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.

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.