Properties of nerve endings with small granular vesicles in the distal colon and rectum of the guinea-pig

The appearance and distribution of nerve endings (varicosities) containing small granular vesicles have been studied in the distal colon and rectum of the guinea-pig with the electron microscope. Two types of varicosity were recognised. They were distinguished by differences in their synaptic vesicles and in their distribution in the layers of the gut wall. The first type resembled noradrenergic nerves in having predominantly (92%) small vesicles and few (8%) large granular vesicles (90 nm diameter). This type was common in the plexuses and at the medial-adventitial border of arteries and arterioles but was sparsely distributed in the muscle coat. The second type had a lower proportion of small vesicles (69%) and a higher proportion (31%) of large granular vesicles (132 nm diameter). This type was absent in Auerbach's plexus, well represented in the muscle coat and Meissner's plexus and not associated with blood vessels. The first type was labelled with 5-hydroxydopamine, a specific marker for noradrenergic nerves, and disappeared after extrinsic denervation. a procedure which causes degeneration of noradrenergic nerves in the gut. The second type was unaffected by 5-hydroxydopamine and extrinsic denervation.It is concluded that the two types of small granular vesicle-containing varicosities belong to different axons and that the first type is noradrenergic. The second type of nerve axon has not been described in the gut before and is intrinsic to it. From the distribution and numbers of these axons in the circular muscle it would seem that they play an important role in gut motility.     

Ultrastructural identification of noradrenergic axons and their distribution within the enteric plexuses of the guinea-pig small intestine

Summary Noradrenergic axons in the enteric plexuses of the guinea-pig ileum have been identified at the ultrastructural level using three techniques: the chromaffin reaction, localization of dopamine--hydroxylase (DBH) with horseradish peroxidase-conjugated antibody, andin vivo andin vitro loading with 5-hydroxydopamine (5-OHDA).In the myenteric (Auerbach's) plexus from normal ileum all of these methods produced electron-dense deposits in a distinctive population of axonal varicosities that contained many flattened vesicles (usually more than 30% of the total number of vesicles), as well as oval or irregularly shaped vesicles. When noradrenergic axons to the small intestine had degenerated after surgical denervation, no profiles containing vesicles with electron-dense deposits were observed with the chromaffin reaction, DBH localization or loading with 5-OHDA. Pretreatment with 6-hydroxydopamine (6-OHDA) substantially reduced the number of noradrenergic axons identified by these three techniques. Axons with many flattened vesicles of similar dimensions but without dense cores were found in myenteric plexus from conventionally fixed intestine. These axons had the same distribution within the ganglia as cytochemically labelled noradrenergic terminals and disappeared after extrinsic denervation.In the normal submucous (Meissner's) plexus, both 5-OHDA loading and the chromaffin reaction produced electron-dense granules in small and large vesicles in some axon terminals. In ganglia labelled by these techniques, reactive terminals contained many small round vesicles and few flattened and large round vesicles as did a population of nonreactive terminals. In axon terminals of submucous plexus labelled with anti-DBH, flattened vesicles were found to be more numerous than with the other treatments. As in the myenteric plexus, all reactive axons disappeared from the submucous plexus after extrinsic denervation. In conventionally processed submucous ganglia, noradrenergic axon profiles could not be distinguished from some non-noradrenergic profiles on the basis of types and proportions of vesicles.In the myenteric plexus noradrenergic axon terminals were seen most often near the edges of ganglia. Noradrenergic varicosities also occurred near nerve cell bodies but were rarely found in internodal strands. In the submucous plexus noradrenergic terminals appeared to be randomly distributed throughout submucous ganglia. No axosomatic synapses formed by noradrenergic axons were found in either plexus, but synapses on nerve processes were occasionally encountered in submucous ganglia.     

On certain fluorescent axon terminals containing granular synaptic vesicles in the cerebellar nucleus lateralis

Summary Thin, unmyelinated, green fluorescent fibers bearing fine beads or varicosities have been found in the neuropil and near blood vessels of the nucleus lateralis. In electron micrographs these fibers are identifiable as a class of axons, the CAT fibers, which contain large and small granular synaptic vesicles and agranular vesicles in their varicosities. There are two types of CAT fiber. 1) The CAT₁ terminals contain many large and elongated vesicles, 700–1700 Å in size, with dark, homogeneously dense centers; a few small granular vesicles each with an intensely osmiophilic particle, and small agranular vesicles. These terminals have not been seen in synaptic contact with other elements of the neuropil. 2) The CAT₂ terminals have a very thin unmyelinated connecting thread between small varicosities. The varicosities contain small agranular synaptic vesicles and small granular ones containing either a single dense particle, or an elliptical, intensely osmiophilic droplet flanked by lighter semicircular particles. Large granular vesicles, 750–950 Å each, with a variably dense center, are also found. These terminals form conventional axodendritic synapses with Gray's type 1 synaptic junctions and the subsynaptic specialization of Taxi, as well as synapses on thorns of spiny neurons.It is suggested that the CAT₁ and CAT₂ fibers may be the electron microscope equivalents of norepinephrine- and 5-hydroxytryptamine-containing fluorescent axons. These fibers probably have extrinsic origins since no fluorescent cells or perikarya with small or large granular vesicles have been found in the lateral nucleus. Their origins, however, are unknown. The proximity of these fluorescent fibers to blood vessels is discussed, and their functions are the subject of some speculation.Supported in part by U.S. Public Health Service grants NS10536, NS03659, Training grant NS05591 from the National Institute of Neurological Diseases and Stroke and a William F, Milton Fund Award from Harvard University.     

Uptake and retention of [3H]adrenaline by central monoaminergic neurons: a light- and electron-microscope radioautographic study after intraventricular administration in the rat

Paraventricular and paracisternal regions of adult rat central nervous system were investigated by light- and electron-microscope radioautography after intraventricular administration of tritiated adrenaline. In tissue primarily fixed by glutaraldehyde perfusion and post-fixed by immersion in osmium tetroxide, there were no aggregates of silver grains indicative of intraneuronal accumulation of the tracer, except over perivascular nerve terminals at the base of the brain. In contrast, when both fixation and postfixation were carried out by rapid vascular perfusion, preferentially labeled nerve cell bodies and axonal varicosities (i.e. terminals) were detected in various anatomical areas known to contain dopaminergic and/or noradrenergic neurons. Serotoninergic axonal varicosities in the supraependymal plexus and subcommissural organ, as well as a small group of nerve cell bodies of undetermined chemical identity in the n. paraventricularis thalami were also found to be labeled. Addition of a ten-fold higher concentration of non-radioactive serotonin to the solution of [3H]adrenaline suppressed the reactivity in the subcommissural organ and the supraependymal plexus but had no such effect elsewhere in brain. Lesioning of the nigrostriatal dopaminergic system with 6-hydroxydopamine prior to [3H]adrenaline injection eradicated axon terminal labeling in the ipsilateral neostriatum. Electron-microscopic examination of [3H]adrenaline-labeled varicosities in the neostriatum, lateral septum, arcuate nucleus and median eminence extended earlier observations on the ultrastructure of the catecholaminergic innervation of these regions. It was concluded that both dopaminergic and noradrenergic neurons as well as certain serotonin-containing axon terminals can take up and retain [3H]adrenaline, although they probably have lesser affinity for this amine than for their own transmitter. Due to the fact that presumptive adrenergic neurons are intermingled with dopaminergic and noradrenergic elements, further work will be needed to determine to which extent they also contributed to [3H]adrenaline uptake in the present experimental conditions.     

The ultrastructure of Auerbach's plexus in the guinea-pig. II. Non-neuronal elements

Summary The ultrastructural features of non-neuronal cells associated with Auerbach's plexus in the stomach, ileum and colon of the guinea-pig have been examined. Apart from Schwann, mast and interstitial or fibroblast-like cells, two other cell types are described that do not appear to have been reported previously. Of these two cell types, one was found external, but close to, the plexus and contained large granular vesicles. The other cell type contained numerous glycogen-like granules, was situated close to or within axon bundles and had processes that extended within and peripheral to nerve bundles as well as being close to smooth muscle cells. Although axon varicosities were opposed to both the processes and cell body of the second type of cell, synaptic-like contacts were not observed.     

The neurochemistry and innervation patterns of extrinsic sensory and sympathetic nerves in the myenteric plexus of the C57Bl6 mouse jejunum

In vitro anterograde tracing of axons in mesenteric nerve trunks using biotinamide in combination with immunohistochemical labelling was used to characterize the extrinsic nerve projections in the myenteric plexus of the mouse jejunum. Anterogradely-labelled spinal sensory fibres innervating the enteric nervous system were identified by their immunoreactivity for calcitonin gene-related peptide (CGRP), while sympathetic noradrenergic fibres were detected with tyrosine hydroxylase (TH), using confocal microscopy. The presence of these markers has been previously described in the spinal sensory and sympathetic fibres. Labelled extrinsic nerve fibres in the myenteric plexus were identified apposing enteric neurons that were immunoreactive for either calretinin (CalR), calbindin (CalB) or nitric oxide synthase (NOS). Of the total anterogradely labelled axons in the myenteric plexus, 20% were CGRP-immunoreactive. Labelled CGRP-immunoreactive varicosities were closely apposed to CalR-immunoreactive myenteric cells, many of which were Dogiel type I (40%; interneurons) or type II (20%; intrinsic sensory) neurons. Labelled CGRP-immunoreactive varicosities were also observed in close appositions to CalB-immunoreactive myenteric cell bodies, of which a small subset had type II morphology (18%; intrinsic sensory neurons). A further 43% of all biotinamide-filled fibres were immunoreactive for TH and these fibres were apposed to CalR-immunoreactive cell bodies (small-sized; excitatory motor neurons) and NOS-immunoreactive cell bodies (either type I or small neurons; inhibitory motor neurons and interneurons) in the myenteric plexus. The results provide a neurochemical and neuroanatomical basis for connections between dorsal root afferent neurons and myenteric neurons and suggest an anatomical substrate for the well-known modulation of enteric circuits from sympathetic nerves. No anterogradely-labelled fibres were stained for NOS-immunoreactivity, despite more than 60% of dorsal root ganglion (DRG) neurons retrogradely labelled from the jejunum showing NOS-immunoreactivity. This was due to a substantial, time-dependent, and apparently selective, loss of NOS from extrinsic axons under in vitro conditions. Lastly, a small population of non-immunoreactive biotinamide-filled fibres (<1%) gave rise to dense terminal structures around individual myenteric cell bodies lacking CalR, CalB or NOS. These specialized endings may represent vagal fibres or a subset of spinal sensory neurons that do not contain CGRP.     

Serotonergic terminals in the neural sheath of the blowfly nervous system: electron microscopical immunocytochemistry and 5,7-dihydroxytryptamine labelling

With serotonin immunocytochemistry we have demonstrated an extensive plexus of immunoreactive varicose fibres in the neural sheath of the nervous system of the blowfly, Calliphora. These fibres are located in the neural sheath of the following regions: the maxillary-labial and labrofrontal nerves of the cerebral ganglia, the cervical connective, the dorsal surface of the thoracicoabdominal ganglia, two pairs of prothoracic nerves and the median abdominal nerve. We identified the serotonin-immunoreactive neural processes in the electron microscope by means of the peroxidase-antiperoxidase method. Immunoreactivity was seen in large granular vesicles (ca 100 nm), on membranes of smaller (ca 60 nm) and larger (ca 100 nm) agranular vesicles, along the inner surface of the axolemma, along neurotubules and outer membranes of mitochondria. By conventional electron microscopy we found numerous varicose neural processes in the neural sheath of some of the above regions. These varicosities are of at least two types. One type corresponds to the serotonin-immunoreactive profiles. A second type contains large granular vesicles (ca 200 nm) of variable electron density. 5,7-Dihydroxytryptamine injected into the head capsule labelled varicosities in the neural sheath, corresponding to the ones identified with serotonin immunocytochemistry. The electron-dense labelling was seen in flattened vesicles within these varicosities. We propose that the serotonin-immunoreactive fibers in the neural sheath constitute neurohemal regions for the release of serotonin into the circulation. The finding of another morphological type of varicose fibers in the neural sheath suggests the presence of further putative neurohormones in these regions.     

Axon terminals of the intrinsic neurons in the nucleus lateralis of the cerebellum an electron microscope study

Summary The large projection neurons of the lateral nucleus have long axons, which leave the cell mass in the superior cerebellar peduncle. These axons emit myelinated recurrent collaterals which have synaptic varicosities en passant. The varicosities are 2–5 m in diameter and contain round, agranular synaptic vesicles ranging between 280 and 480 Å with diameters of approximately 400 Å. The vesicles lie in a moderately dark axoplasmic matrix with a mean packing density of 281/m². The varicosities synapse through Gray's type 1 junctions with dendrites and thorns of large and small neurons. They constitute 22% of the total axonal population on dendrites of large neurons and 10% on dendrites of small neurons. The recurrent collateral system may provide a means for positive feedback to the same neuron and other neurons of the neuropil.The small neuron or interneuron has a short axonal plexus. The axon is myelinated, and is distinctive with a light axoplasmic matrix and varicosities containing elliptical synaptic vesicles. The vesicles are loosely dispersed with a mean population density of 44/m². These varicosities synapse through an intermediate type of junction upon the somata of certain large and small neurons and they consitute 14% and 22% of the axosomatic synapses respectively. They also make synapses on dendrites, constituting 12% and 25% of the total population of axons synapsing with dendrites of large neurons and those of small neurons respectively. It is suggested that these are the inhibitory interneurons of the lateral nucleus.The corticonuclear input through Purkinje axons is the dominant influence on the lateral nucleus neurons. This inhibitory input is considerably larger on the large neurons than on the small ones. It is speculated that the axosomatic synapses are inhibitory. Excitatory influences, through the collaterals of mossy and climbing fibers and the recurrent collaterals of the large intrinsic neurons, impinge upon the dendrites, where the axons of both Purkinje cells and interneurons also terminate.Supported in part by U.S. Public Health Service grants NS10536, NS03659, Training grant NS05591 from the National Institute of Neurological Diseases and Stroke, and a William F. Milton Fund Award from Harvard University.     

The ultrastructure of Auerbach's plexus in the guinea-pig. I. Neuronal elements

Summary The ultrastructural features of nerve cell bodies and axon profiles within Auerbach's plexus in the stomach, ileum, caecum and colon of the guinea-pig have been examined. Nerve cell bodies have been tentatively classified into nine different types according to their size, distribution of organelles, location and relationship to satellite cells. Except for cell size, no attempt has been made to correlate ultrastructural with light microscopical observations.On the basis of vesicular size, shape and content, eight morphologically distinct types of axon profile have been identified as well as two profile types which are thought to reflect different physiological conditions. The axons contained various populations of small, mostly granular vesicles; small, round agranular vesicles; small, flattened vesicles; large flattened or elongated vesicles; and three types of large vesicle with granular contents distinguished by size. Some correlation between types of axon profile and two types of nerve cell body was recognized. However, more than one type of axon profile usually formed synapses with one type of cell body, and a precise correlation was not determined.     

On the identification of the afferent axon terminals in the nucleus lateralis of the cerebellum an electron microscope study

Summary Axon terminals in the neuropil of the lateral nucleus can be divided into six classes, each with a specific constellation of characteristics that consistently occur together. Two of these classes have synaptic varicosities with elliptical synaptic vesicles, one in a dense, the other in a sparse matrix, and both make axosomatic and axodendritic synapses. The remaining four classes all have round synaptic vesicles and do not make axosomatic synapses. In the first of these four, the vesicles are tightly packed in a dense matrix, in another they are loosely dispersed, and in the third they are clustered. In the fourth, large granular vesicles predominate. Of these six classes, the most numerous belong to the axons of the Purkinje cell terminal arborization. These boutons resemble their counterparts in the cerebellar cortex, the recurrent collaterals of the Purkinje axon. They have elliptical and flat synaptic vesicles in a dark matrix. The varicosities terminate on somata and dendrites of large and small neurons and constitute the majority of their input. Purkinje axons constitute 86% of the total population of terminals on large neuronal perikarya and 50% of those on their dendrites, but only 78% on the somata of small neurons and 31% on their dendrites. The terminals of climbing fiber collaterals are recognized by their resemblance in electron micrographs to the terminals of the climbing fiber arborization in the cerebellar cortex. They bear round synaptic vesicles packed into a dense axoplasmic matrix and make Gray's type 1 axodendritic synapses with large and small neurons. These axons are restricted to the lateral and ventral aspects of the nucleus and constitute 5% of the terminals on large cell dendrites and 6% of those on small neurons. The axons tentatively identified as collaterals of mossy fibers are myelinated fibers with a light axoplasm containing round synaptic vesicles, dispersed throughout their varicosities. They make Gray's type 1 synapses and constitute a fair percentage of the total axodendritic contacts in the neuropil, 22% on large neurons and 28% on small neurons. The bases for these tentative identifications are discussed in detail, as are the various synaptic relationships undertaken by each class of axon. The remaining 4 classes of axons of the neuropil will be described in subsequent papers.Supported in part by U.S. Public Health Service grants NS 10536 and NS 03659, Training grant NS 05591 from the National Institute of Neurological Diseases and Stroke, and a William F. Milton Fund Award from Harvard University.     

An ultrastructural analysis of the developing enteric nervous system of the guinea-pig small intestine

Summary The developing enteric nervous system of the guinea-pig has been analysed ultrastructurally. In addition, electron microscope autoradiography, following incubation with tritiated 5-hydroxytryptamine ([³H]5-HT) or tritiated norepinephrine ([³H]NE) was used to locate the developing axons of enteric serotoninergic and adrenergic neurons respectively. Observations have been correlated with previous studies of the development of the various types of enteric neuron and the onset of intestinal neuromuscular function. Prior to 25 days of gestation no neurons can be recognized morphologically. Neurons first appear at 25 days' gestation, together with a primitive neuropil in neural islands within the outer gut mesenchyme. Ganglion cell precursors are primitive at first and resemble the cells in the surrounding mesenchyme. Growth cones are abundant but there are no terminal varicosities or synapses. The circular muscle also begins to form at this time. At 32 days' gestation the longitudinal layer of smooth muscle can be discerned and, within the myenteric plexus, terminal axonal varicosities appear containing small (about 50 nm in diameter) electron-lucent synaptic vesicles. The submucosal plexus appears to be derived from neurons and neurites that reach it from the earlier-developing myenteric plexus. The submucosal plexus can be recognized at 38 days of gestation but is not well developed until day 42. Synapses on ganglion cell somata first appear in the myenteric plexus on gestational day 38 and are numerous on day 42 when the first axo-dendritic synapses can be seen. Between days 42 and 48 the developing neural tissue and growing smooth muscle cells interdigitate but after day 48, the plexus becomes ensheathed by supporting cells and connective tissue and this interdigitation is lost. Prior to day 48 most varicosities contain small electron-lucent synaptic vesicles; however, after this time a variety of terminals appears. Between days 48 and 53 of gestation evidence of degenerating neuronal processes is common, indicating that cell death may occur. Electron microscopic autoradiography with [³H]5-HT reveals labelling of axons in the neuropil of the myenteric plexus at day 32 of gestation. Some primitive appearing cell bodies, however, are also labelled and these cells seem to be entering the myenteric plexus from the surrounding mesenchyme. After 42 days of gestation [³H]5-HT labels only axons of both nerve plexuses. Often, labelled terminals are apposed to ganglion cells or dendrites. In contrast, significant labelling by [³H]NE is not found until gestational day 48. Axons are labelled by [³H]NE and these tend to be located at the interface between the myenteric plexus and the surrounding connective tissue.     

A quantitative study of the effects of age on the noradrenergic innervation of Auerbach's plexus in the rat

Noradrenergic nerves were demonstrated in stretch preparations of Auerbach's plexus and longitudinal muscle from the proximal jejunum of Wistar rats using glyoxylic acid-induced fluorescence. The density of the noradrenergic nerve plexus and the number of nerve terminal varicosities/frame area were assessed using a Quantimet 800 image analyser and the number of varicosities/unit length of nerve was measured manually with a calibrated planimeter. With increasing age, especially between 12 and 18 months there occurs a breakdown of plexus regularity and noticeably reduced levels of axonal fluorescence. Image analysis showed a decrease in the total area of the plexus of more than 50% and a decrease of almost 75% in the total number of varicosities. The frequency of varicosities per 100 micron of axon decreased from 18.79 at 12 months to 14.79 at 18 months. Significant changes in these parameters did not occur during the following 6 months. The dramatic decrease in the density of the noradrenergic innervation of Auerbach's plexus and the fall in number of varicosities with age implies a reduction in the potential of the sympathetic nervous system to influence control over motility of the jejunum in the aged rat.     

Immunohistochemical studies of the enteric nervous system in tissue culture and in situ: localization of vascoactive intestinal polypeptide (VIP), substance-P and enkephalin immunoreactive nerves in the guinea-pig gut

Immunofluorescence methods have been used to determine the detailed distribution of vasoactive intestinal polypeptide (VIP), substance-P and enkephalin nerve fibres in fixed cryostat sections from guinea-pig duodenum, jejunum, ileum, caecum at the site of the taenia coli, and proximal and distal colon. A novel method is used involving immunostaining of tissue culture preparations of both myenteric and submucous plexuses. These preparations allow each plexus to be studied in isolation from all axonal input for the first time, since they provide unequivocal extrinsic denervation together with severance of any intrinsic connections between the plexuses. In tissue sections the most prominent sites of VIP and substance-P immunoreactive fibres are the ganglia of the myenteric and submucous plexuses, the circular muscle layer and the longitudinal muscle of the taenia coli. In addition, VIP is prominent in the lamina propria of the submucosa except in the caecum. Enkephalin-immunopositive fibres are restricted to the ganglia of the myenteric plexus, the circular muscle layer and the longitudinal layer of the taenia coli. The culture preparations reveal that intrinsic ‘VIP neurons’ are common in the submucous plexus of the caecum and colon. They are also present, but in much lower numbers, in the myenteric plexus of the small intestine and colon but are not found in the myenteric plexus of the caecum. Intrinsic ‘substance-P neurons’ are present in the myenteric plexus from the small intestine, caecum and colon as well as in the submucous plexus of the colon; intrinsic ‘substance-P neurons’ are not found in the submucous plexus of the caecum. ‘Enkephalin neurons’ are numerous in the myenteric plexus of the small intestine, caecum and colon but are absent from the submucous plexus. Immunoreactivity is compared in the normal and denervated caecum by both the histochemical method and by radioimmunoassay of tissue extracts. In conjunction with the studies on tissue cultures, the results provide evidence for intrinsic reciprocal connections between the myenteric and submucous plexus of the caecum by neurons containing VIP and substance-P.An extensive comparison of these results with data from functional studies shows that the distribution of VIP, substance-P and enkephalin fibres in the gut is broadly in agreement with present knowledge of the action of these peptides on gut tissue, if it is assumed that they function as neurotransmitters or neuromodulators. In some instances, however, peptide-containing fibres and pathways are found which do not correlate with present knowledge obtained from functional studies. These observations provide new clues to the role of peptide neurons in gut function.     

Opioid neurons and pain modulation: an ultrastructural analysis of enkephalin in cat superficial dorsal horn

To clarify the circuitry through which opioid compounds modulate spinal and trigeminal nociceptive transmission, we have examined the synaptic associations formed by leucine-enkephalin-containing (enkephalin) neurons in the superficial dorsal horn of the cat. As described previously, punctate enkephalin immunoreactivity is concentrated in the marginal layer (lamina I) and in both the outer and inner layers of the substantia gelatinosa (lamina IIo and IIi). In colchicine treated cats, enkephalin perikarya are most numerous in lamina I and at the border between laminae I and II. Ultrastructural analysis reveals that enkephalin cells receive a diverse afferent input. The majority of afferent inputs are presynaptic to the enkephalin dendrites; few axosomatic synapses are seen. Among these presynaptic axonal profiles are unlabeled axons which resemble primary afferent terminals, including the characteristic central axonal varicosity. Enkephalin dendrites are also postsynaptic to enkephalin immunoreactive axons. Two types of enkephalin axonal profiles appear in the superficial dorsal horn. Class I profiles are only found in lamina I. These are large profiles which form few synapses; those synapses made are axodendritic. Class II enkephalin axons are smaller and are distributed in both layers I and II. While Class II axons most commonly form axo-dendritic synapses, they also form axo-axonic synapses with flat vesicle-containing profiles; the latter are generally presynaptic to the enkephalin terminals. Serial analysis further revealed that both the enkephalin and the flat vesicle-containing profile synapse onto a common dendrite. Although enkephalin axons frequently lie adjacent to round vesicle-containing profiles, anatomical evidence that opioid axons form synapses with this type of ending was not found. An additional type of enkephalin vesicle containing-profile is found in layer IIi; its morphological features do not clearly distinguish its axonal or dendritic origin. These endings are typically postsynaptic to unlabelled central endings, and provide minimal presynaptic input to other elements in the neuropil. Like some class II axons, these labelled profiles contain vesicles which cluster at the membrane immediately adjacent to unlabelled central axons. These results indicate that spinal enkephalin neurons receive a variety of synaptic inputs. These include inputs which may derive from primary afferent axons. Enkephalin neurons, in turn, influence nociceptive transmission predominantly through postsynaptic mechanisms. Finally, while we did not observe enkephalin terminals presynaptic in an axoaxonic relationship, the possibility that enkephalin neurons modulate the excitability of fine fiber nociceptive and nonnociceptive afferents via "nonsynaptic interactions" is discussed.     

Glomerular handling of immune complex in the acute phase of active in situ immune complex glomerulonephritis employing cationized ferritin in rats

The distribution and co-localization of nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) were examined by means of immunohistochemistry and NADPH diaphorase (NADPH-d) histochemistry in the gut of patients with Hirschsprung's disease. In the normoganglionic segment, many nitrergic nerve cells were localized in Auerbach's plexus and nerve fibres were observed preferentially in the circular muscle. The submucosal nitrergic nerve cells were mainly situated in Schabadasch's plexus with occasional cells demonstrable in Meissner's plexus. NOS and VIP were co-localized in most ganglion cells of Auerbach's plexus. In the oligoganglionic segment, a marked reduction of NOS- and VIP- positive nerve cells and fibres was noticed in both the myenteric and submucosal plexuses, and nitrergic fibres had disappeared in the inner layer of the circular muscle. In the aganglionic segment, NOS and VIP were revealed only in extrinsic nerve fasciculi and rami and co-localized in a few fibres. From these observations, the inner layer of the circular muscle of the oligoganglionic segment and the whole of the muscularis propria of the aganglionic segment were considered to be totally lacking in nitrergic innervation. Nitrergic nerves of the human colon comprise both intrinsic and extrinsic elements and the majority of intrinsic nitrergic nerve cells contain VIP. Very low numbers of extrinsic nitrergic fibres contain VIP.     

Immunoelectron microscopic analysis of the synaptic connectivity of serotoninergic neurons grafted to the 5,7-dihydroxytryptamine-lesioned rat spinal cord.

The connections between the host and 5-hydroxytryptamine-containing neurons grafted to the spinal cord have been analysed using electron microscopic immunohistochemistry. Adult rats with 5,7-dihydroxytryptamine lesions of the brain and spinal cord received implants of embryonic medullary raphé neurons at three sites in the spinal cord. Eight to 10 months after grafting, the transplanted 5-hydroxytryptamine-positive neurons had formed extensive and complex contacts with spines, dendrites, perikarya and vesicle-containing structures in both the dorsal and ventral horns. Reinnervation of laminae IV-VI was less rich. In the graft itself, connections were also made between non-immunoreactive varicosities and 5-hydroxytryptamine-containing dendrites, and somata, but the exact origin of the afferents was not determined. Outside the implant site, no obvious synaptic junctions onto grafted 5-hydroxytryptamine-immunoreactive boutons were obvious, although labelled and unlabelled varicosities were often in close apposition. Synaptic junctions in the dorsal horn were predominantly symmetric, with the presynaptic varicosity containing mostly small agranular vesicles. By contrast, in the ventral horn most junctions were asymmetric, while the presynaptic element contained both small agranular and large dense-core vesicles. The results demonstrate that the types of synaptic contacts formed between the grafted 5-hydroxytryptamine neurons and the host spinal cord are remarkably similar to those found in intact spinal cord. In addition, the division of morphological differences that exists between 5-hydroxytryptamine-containing boutons in the normal dorsal vs ventral horns is also apparent in the transplanted animals. Finally, there appear to be present several anatomical substrates for the regulation by the host of 5-hydroxytryptamine output from the grafted neurons.     

High-resolution radioautographic study of the serotonin innervation of the rat corpus striatum after intraventricular administration of [3H]5-hydroxytryptamine

In the rat striatum, identification of the serotonin-containing nerve endings at the ultrastructural level has been done by radioautography after ventriculodsternal perfusion of [³H]serotonin. Only the ventricular border of the middle part of the nucleus caudatus-putamen (neostriatum) was studied and, in addition, a small dorsal part of the globus pallidus.For every 100 μm²of thin sections of the neostriatum, 0.5 labelled structures were found, which were either axons (14%), preterminal enlargements (21.5%) or nerve terminals (64.5%). Three kinds of labelled nerve endings could be recognized according to the size and shape of their synaptic vesicles, although all contained occasional large granular vesicles. The first type (36%) was characterized by small and often granular, ovoid vesicles mixed with tubular structures of the same diameter (20 nm). The second type (28%) contained crowded spherical vesicles of 45 nm in diameter which were mostly clear. The third type (36%) contained a mixture of both types of synaptic vesicles. The labelled nerve terminals in the neostriatum showed a few synaptic contacts (mean of 14%) which were, however, more numerous in the second type of nerve endings (25–30%) and mainly of the asymmetric type. In the globus pallidus the serotonin-labelled nerve terminals resembled those in the neostriatum but one additional type was observed, which was characterized by only a few typical synaptic vesicles and by numerous large granular vesicles.The observations made in this study indicate that the serotonin-containing varicosities of the striatum are very similar to those described in some other nuclei. They exhibit characteristic large granular vesicles and the synaptic vesicles often have an eccentric tiny granule after administration of exogenous serotonin and/or inhibition of monoamine oxidase. The peculiar type of nerve endings with tubular vesicles described by others in the cerebellum and in the locus coeruleus was also present in the neostriatum. The morphological heterogeneity of serotonin-containing nerve endings within a single nucleus or from one nucleus to another, is discussed in relation to the amine content, the stage of formation of the synaptic vesicles and their origin in different raphe nuclei.     

Architecture and synaptic relationships in the intermediolateral nucleus of the thoracic spinal cord of the rat: HRP labelling, catecholamine histochemistry and electron microscopic studies

Summary The organization of the intermediolateral nucleus (IML) of the thoracic spinal cord was examined using glyoxylic acid-induced fluorescence histochemistry, retrograde horseradish peroxidase (HRP) labelling and electron microscopy. In serial sections of T2, it was found that the distribution of catecholamine nerve terminals was intimately related to the neuronal perikarya of IML. Potassium permanganate fixation and 5-hydroxydopamine treatment revealed small dense-cored vesicles in axon varicosities with or without synaptic specializations. A gelatinous region, composed of small diameter dendrites and unmyelinated axons, formed a narrow longitudinal bundle in the centre of the nucleus. The population of the axon varicosities in the IML was 0.17 ± 0.02/m² in 75 nm sections. The average size of the axon varicosities with flat synaptic vesicles was 1.44 ± 0.05 m² and that of varicosities with spherical vesicles was 0.97 ± 0.02 m². After HRP injection into the superior cervical ganglion, ipsilateral IML neurons were labelled in T1–T3 segments of the spinal cord. Axon varicosities with flat and others with spherical synaptic vesicles synapsed on the dendrites labelled by HRP. Among axon varicosities synapsing on the preganglionic sympathetic neurons, 74.8 ± 7.1% at axo-somatic synapses and 46.0 ± 6.7% at synapses on proximal dendrites contained flat synaptic vesicles.     

Ultrastructural features of a human neuroblastoma cell line treated with retinoic acid

This report examines the morphological changes that occur in a line of human neuroblastoma cells (LA-N-5) following treatment with retinoic acid, in vitro. The results demonstrate that retinoic acid induces pronounced differentiation of these cells. Perikarya aggregate into tight clusters and extend long processes that are frequently fasciculated. Growth cones appear at the ends of these processes. Transmission electron microscopy reveals that after 10 days of treatment these long neurites give rise to varicosities which contain clusters of large dense-core vesicles and smaller clear vesicles. After 18 days of treatment the cultures cease to differentiate further. The pattern of neurite outgrowth is very complex by this point and the frequency of growth cones and vesicle-containing varicosities is greatly increased compared with shorter treatments. Most of these varicosities contain a mix of large dense-core vesicles and smaller clear vesicles and in some profiles the clear vesicles are round while in others they are pleomorphic. Despite this increase in the number of vesicle-containing profiles no membrane specializations were seen that resemble mature synapses. The present results demonstrate that retinoic acid can produce morphological changes in these cells in culture, and that these changes closely mimic those of normal differentiating neurons in culture. Considered with previous studies, these findings suggest that this cell line might provide a useful model system for studying neural differentiation.     

An ultrastructural study of nerve profiles in the myenteric plexus of the rabbit colon

The ultrastructure of the myenteric plexus from the rabbit colon was examined in both conventionally fixed tissue and also material fixed with the chromaffin method. Montages of the ganglia were analysed semi-quantitatively. Six main types of axon profile are described and classified on a morphological consideration of the vesicle population. Most axon types formed synapses with myenteric neurons. Two kinds of chromaffin-positive nerve fibre were seen, one containing a predominance of small granular vesicles, the other containing many flattened vesicles. The difficulties in relating axon profile types to putative transmitters are discussed.