Wide distribution and subcellular localization of histamine in sympathetic nervous systems of different species.

Previous studies have demonstrated that histamine (HA) acts as a neurotransmitter in the cardiac sympathetic nervous system of the guinea pig. The aim of the current study was to examine whether HA widely exists in the sympathetic nervous systems of other species and the subcellular localization of HA in sympathetic terminals. An immunofluorescence histochemical multiple-staining technique and anterograde tracing method were employed to visualize the colocalization of HA and norepinephrine (NE) in sympathetic ganglion and nerve fibers in different species. Pre-embedding immunoelectron microscopy was used to observe the subcellular distribution of HA in sympathetic nerve terminals. Under the confocal microscope, coexistence of NE and HA was displayed in the superior cervical ganglion and celiac ganglion neurons of the mouse and dog as well as in the vas deferens, mesenteric artery axon, and varicosities of the mouse and guinea pig. Furthermore, colocalization of NE and HA in cardiac sympathetic axons and varicosities was labeled by biotinylated dextranamine injected into the superior cervical ganglion of the guinea pig. By electron microscopy, HA-like high-density immunoreactive products were seen in the small vesicles of the guinea pig vas deferens. These results provide direct cellular and subcellular morphological evidence for the colocalization of HA and NE in sympathetic ganglion and nerve fibers, and support that HA is classified as a neurotransmitter in sympathetic neurons.

Nerve cells and synapses in the human foetal hypogastric nerves.

The hypogastric nerves of a human foetus of 220 mm C-R length (23(rd) week) were investigated with an electron microscope. These nerves were composed mainly of bundles of unmyelinated fibres and single myelinated fibres. Small ganglia and single ganglion cells were observed in the hypogastric nerves. Light and dark cells were found among the nerve cells. The two types of cell differed in the number of ribosomes and the amount rough endoplasmic reticulum. In the period of development investigated protosynapses and mature synapses were observed in the hypogastric nerves.

Development of the myelin sheath of the hypogastric nerves in a human foetus aged 23 weeks.

The formation of the myelin sheath of the human hypogastric nerves was studied by electron microscopy in a foetus of 23 weeks of postovulatory age (220 mm C-R length). In the investigated foetus the hypogastric nerves were mainly composed of bundles of unmyelinated fibres. The myelinated fibres were seen to be at different stages of myelination. Well myelinated fibres had thick compact laminated myelin. The number of myelin lamellae on a single fibre was 22.

Anatomy of the pelvic plexus and innervation of the prostate gland.

We have examined the anatomy of the pelvic (inferior hypogastric) plexus in six male cadavers, paying particular attention to gross anatomical landmarks that might aid in locating it and have used immunohistochemistry to study the small branches of the plexus that supply the prostate gland. The pelvic plexus was found two finger breadths lateral to the third anterior sacral foramina, lying deep to a line drawn from third sacral vertebra, the conventional level of the recto-sigmoid junction, and the palpable posterior superior surface of the pubic symphysis. Immunohistochemical staining showed small nerve branches from the pelvic plexus entering the prostate gland and the presence of ganglia within the prostate gland that contained both tyrosine hydroxylase positive and negative neuronal cell bodies. This information may be useful in nerve-sparing surgical procedures and in discussions of the functional implications of perturbations of prostate innervation.

Differential susceptibility to ageing of rat preganglionic neurones projecting to the major pelvic ganglion and of their afferent inputs.

We have analysed age-related changes in the morphology of preganglionic neurones in the lumbosacral spinal cord, labelled following injection of retrograde tracers into the major pelvic ganglion of young adult and aged male rats. We have also examined changes in neurotransmitter-characterised spinal afferent inputs to these neurones, or to the nuclei in which they lie, using light and electron microscope immunohistochemistry. In previous investigations of the major pelvic ganglion, the sympathetic, but not parasympathetic, postganglionic neurones were seen to exhibit age-related changes and the same pattern is seen in the preganglionic neurones. This included an apparent reduction in the numbers of sympathetic preganglionic neurones, and a reduction in the length of their dendrites and the complexity of their branches. Ultrastructural immunohistochemical studies described here reveal significant reductions in the area of synaptic contact made by glutamate-immunoreactive boutons onto the dendrites of sympathetic (but not parasympathetic) preganglionic neurones, while contacts from boutons immunoreactive for glycine or gamma-aminobutyric acid (GABA) were unchanged. There is also a reduction in synaptic contacts received by sympathetic somata from boutons immunoreactive for none of these amino acids. Serotonin-immunoreactive terminals are closely associated with preganglionic autonomic neurones, and these are reduced in number in sympathetic, but not parasympathetic, spinal nuclei of aged rats. However, serial section electron microscopy has so far failed to demonstrate conventional synaptic contacts between serotonergic terminals and the dendrites or somata of the preganglionic autonomic neurones. In young animals, axon terminals immunoreactive for thyrotropin-releasing hormone (TRH) are abundant in all spinal laminae including area X, but in aged animals, such terminals are significantly reduced in number in regions containing preganglionic sympathetic, but not parasympathetic, neurones. These results indicate that the sympathetic preganglionic neuron populations that project to the major pelvic ganglion, and the spinal inputs they receive, show a number of degenerative changes in aged rats which are not seen parasympathetic preganglionic neuronal populations.

The hypogastric and thirteenth thoracic ganglia of the rat: effects of age on the neurons and their extracellular environment.

Morphometric analyses of the neurons and microvessels of perfusion-fixed hypogastric (HG) and 13th thoracic (T13) ganglia have been performed in male Wistar rats aged 4, 24 and 30 mo. Estimations of HG volume employing the Cavalieri principle have also been performed and showed that the size of the aged HG is increased by 42%. Routine histological staining of the ganglia with Masson's trichrome indicated that this may be due to the increased amount of interstitial connective tissue which was apparent in the aged animals. The number of neurons per unit area progressively decreased by 38% between ages 4 and 24 mo and by 16% between ages 24 and 30 mo in the HG and by 25% (4 and 24 mo) and 2% (24 and 30 mo) in the T13 ganglion. The total number of neurons in the HG however, estimated by a physical disector analysis, was constant with age. The number of microvessels per unit area, microvessel diameter, neuronal and nuclear areas did not differ significantly between the 3 age groups studied. This observed increase in ganglionic volume and decrease in neuronal packing density may be associated with changes in the extracellular matrix, in particular in glycosaminoglycans whose presence was indicated by metachromasia of the ganglia with toluidine blue. The extracellular matrix was therefore characterised using a panel of monoclonal antibodies against glycosaminoglycans and laminin. Chondroitin-6 sulphate and chondroitin-4 sulphate were present in the interstitial connective tissue, and there was an increase in the expression of both these epitopes at 24 mo, noteably surrounding neuron cell bodies. The expression of chondroitin-4 sulphate/dermatan sulphate was unchanged, thus implying a decreased expression of dermatan sulphate with age. Keratan sulphate and the native chondroitin sulphate epitopes were absent from the ganglia at both ages. Laminin expression was increased in the aged ganglia. It is therefore clear that the constituents of the extracellular matrix are not constant throughout the adult lifespan and that the extracellular matrix may influence neuronal survival in old age. This is the first report characterising age-related changes in the extracellular matrix of autonomic ganglia.

Conduction velocity distribution of afferent fibers in the female rat hypogastric nerve.

Conduction velocities of single afferent fibers in the female rat hypogastric nerve were measured by stimulating dorsal rootlets and recording from the hypogastric nerve. A total of 344 units were identified and measured. They were distributed among dorsal roots T13 to L3 ipsilaterally (75%) and between L1 and L2 contralaterally (25%). Over 95% were found in the L1 plus L2 dorsal roots. Ninety-six percent of the units had conduction velocities less than 2 m/s; the average conduction velocity was 0.98 m/s. By way of contrast, afferents in the postganglionic nerves innervating the urinary bladder with conduction velocities less than 2 m/s constituted 65% of the afferents. We conclude that the overwhelming majority of afferents in the female rat hypogastric nerve are unmyelinated C-fibers.

Met5-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in the pelvic ganglion of the male rat: a light and electron microscopic study.

By using both light and electron microscopic immunocytochemical methods, Met5-Enkephalin-Arg6-Gly7-Leu8 (MEAGL)-like immunoreactive structures were detected in the pelvic ganglion of male rats. Denervation studies were carried out to determine the origin of these immunoreactive fibers and the projection of immunoreactive neurons within the pelvic ganglion. MEAGL-like immunoreactivity was found in numerous axon boutons, some small, intensely fluorescent (SIF) cells, and a few principal ganglion neurons. Most of the immunoreactive nerve fibers formed pericellular plexuses surrounding the ganglion cells. In addition, there were a few scattered varicose fibers. These fiber plexuses could be classified into two types: type I (approximately 90% of fibers), which consisted of 80-120 small boutons that synapsed on either the dendrites (80% of cases) or somata (20% of cases) of principal neurons; and type II (approximately 10% of fibers), which consisted of 20-40 larger boutons that formed axodendritic synapses exclusively. After transection of the hypogastric and pelvic nerves, virtually all of the pericellular fiber plexuses disappeared, whereas the scattered varicose fibers remained. According to their ultrastructure, these remaining fibers were considered to arise from SIF cells. Following the injection of Fast Blue into the bladder wall, some of the MEAGL-like immunoreactive principal neurons were retrogradely labeled. The results of this study indicate that there are two origins for the MEAGL-like immunoreactive fibers detected in the pelvic ganglion: most arise from preganglionic neurons in the spinal cord, and a small proportion may originate from intraganglionic MEAGL-like immunoreactive SIF cells or principal neurons. Some MEAGL-like immunoreactive principal neurons may project to the urinary bladder.

Modifications of embedding tissues in durcupan ACM (Fluka) based on viscosity index measurements.

Viscosity of Durcupan ACM mixtures without as well as with accelerator was measured, at temperatures of 25, 40, and 50 degrees C respectively. The time course of polymerization was expressed within first few hours following admixture of accelerator, at temperature mentioned above. On the basis of our findings a modified embedding schedule for tissues (peripheral autonomic nerves and ganglia) has been recommended: infiltration in Durupan 1 (without accelerator) could be performed at 50 degrees C, when viscosity of embedding mixture is greatly reduced, to 190 mPa. s. Further infiltration in Durcupan 2 (with accelerator added) is carrier out at 40 degrees C; the initial viscosity of embedding medium at this temperature is 440 mPa. s. and even after 4 hours it keeps below 200 mPa. s. So the infiltration time could be prolonged in contrast to that at 50 degrees C, which usually brings better embedded blocks and better ultrathin sections.

The uptake and retrograde transport of horseradish peroxidase--polylysine conjugate by ligated postganglionic sympathetic nerves in vitro.

The uptake and retrograde transport of horseradish peroxidase (HRP) and horseradish peroxidase-poly-L-lysine conjugate (HRP-PL) were compared using a system comprising the guinea-pig inferior mesenteric ganglion (IMG) and ligated hypogastric hypogastric nerves maintained in vitro in a twin chamber apparatus. 0.5 mg of HRP-PL applied to the ligated nerves produced stronger retrograde labelling of neurons within the IMG than did 10 mg of HRP. This may have been due to the greater uptake of HRP-PL in a vesicular form by the axons immediately proximal to the ligation. The possible roles of large rounded vesicles and elongated cisternae in retrograde axoplasmic transport are discussed.

[Effect of colchicine and lumicolchicine on the ultrastructure of non-myelinated axons in autonomic nerves in cat in vitro]. / Die Wirkung des Kolchicins und Lumikolchicins auf die Feinstruktur markloser Axone in autonomen Nerven der Katze in vitro

The effects of colchicine and lumicolchicine on the ultrastructure of non-myelinated axons in cat autonomic nerves were studied using in vitro preparations of inferior mesenteric ganglion/hypogastric nerves. After 24 hrs of in vitro incubation with colchicine added to the medium (10 microng/ml) a significant decrease in number of neurotubules per 1 axon was observed. In the presence of a solution of alpha-beta and gamma-lumicolchicine (10 microng/ml) severe degenerative changes occured in axons and Schwann cells. At a lower dose of lumicolchicine (3 microng/ml) these changes were less frequent and the number of neurotubules per 1 axon did not differ from that in control nerves.

The major ganglion in the pelvic plexus of the male rat: a histochemical and ultrastructural study.

To further evaluate the role of autonomic ganglia in the regulation of pelvic visceral activity, the neural elements in the major pelvic ganglion of the male rat have been studied with histochemixal and electron microscopic techniques. The principal findings are that the ganglion is composed of cholinergic and adrenergic ganglion cells as well as small intensely fluorescent (SIF) cells. Polarity in the ganglion is indicated by clustering of small ganglion cells which stain intensely for acetylcholinesterase (AChE) along the pelvic nerve while larger cells, with weak to moderate AChE activity, collect near small branches of the hypogatrric nerve. Some cholinergic ganglion cells are enclosed by a plexus of adrenergic terminals. SIF cells appear to be in contact with both cholinergic and adrenergic cells, although many of the fluorescent beads around adrenergic neurons may be short dendrites of ganglion cells, rather than processes of SIF cells. Two types of SIF cells may be distingiosjed on the basis of size and morphology of their granulated vesicles. Afferent synapses of the cholinergic type were common on SIF cells of the large granule and small granule type. Portions of SIF cells with large granules occur within the capsule of ganglion cells. Contacts seen here were interpreted as efferent synapses from SIF cells to the dendrites of ganglion cells.

Two populations or granular vesicles in constricted post-ganglionic sympathetic nerves.

1. A comparative study has been made of the effects of different fixatives on the ultrastructural appearance of granular vesicles accumulated against a constriction in cat hypogastric nerves. 2. After fixation in either osmium tetroxide or in glutaraldehyde followed by post-osmication accumulations of large granular vesicles (65-90 nm in diameter) were observed in profiles of swollen axons. 3. Fixation in acrolein and sodium dichromate revealed a second population of small granular vesicles (mostly 30-50 nm in diameter) in addition to the large vesicles seen after the other fixatives. 4. In reserpinized cats the small granular vesicles were absent. The large granular vesicles were much less numerous, irrespective of the fixative used. 5. It is suggested that either the small vesicles occur in normal noradrenergic axons but are only revealed by catecholamine-sensitive fixatives or they occur as a result of constricting the nerve trunk. Evidence for and against these possibilities is discussed.