Morphological and biochemical studies on the development of cholinergic properties in cultured sympathetic neurons. I. Correlative changes in choline acetyltransferase and synaptic vesicle cytochemistry

Under certain culture conditions, neonatal rat superior cervical ganglion neurons display not only a number of expected adrenergic characteristics but, paradoxically, also certain cholinergic functions such as the development of hexamethonium-sensitive synaptic contacts and accumulation of choline acetyltransferase (ChAc). The purpose of this study was to determine whether the entire population of cultured neurons was aquiring cholinergic capabilities, or whether this phenomenon was restricted to a subpopulation. After 1--6 and 8 wk in culture, neurons were fixed in KMnO4 after incubation in norepinephrine and prepared for electron microscopy analysis of synaptic vesicle content to determine whether vesicles were dense cored or clear. ChAc, acetylcholinesterase (AChE), and DOPA-decarboxylase (DDC) activities were assayed in sister cultures. In the period from 1 to 8 wk in culture, the average ChAc activity per neuron increased 1,100-fold, and the DDC and AChE activities increased 20- and 30-fold, respectively. After 1 wk in culture, 48 of 50 synaptic boutons contained predominantly dense-cored vesicles, but by 8 wk the synaptic vesicle population was predominantly of the clear type. At intermediate times, the vesicle population in many boutons was mixed. The morphology of the synaptic contacts on neuronal surfaces was that characteristic of autonomic systems, with no definite clustering of the vesicles adjacent to the area of contact. Increased vesicle size correlated with increasing age in culture and the presence of a dense core. Considering these data along with available physiological studies, we conclude that these cultures contain one population of neurons that is initially adrenergic. Over time, under conditions of this culture system, this population develops cholinergic mechanisms. That a neuron may, at a given time, express both cholinergic and adrenergic mechanisms is suggested by the approximately equal numbers of clear and dense-cored vesicles in the boutons found at the intermediate times.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus

Summary Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diamin-obenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus. Dual electron microscopic immunolabeling

Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diaminobenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

An Ultrastructural Account of Otoplanid Turbellaria Neuroanatomy I. The cerebral ganglion and the peripheral nerve net

The otoplanid nervous system investigated in Otoplana truncaspina Lanfranchi, 1969 and Parotoplanella heterorhabditica Lanfranchi, 1969 consits of: (a) an ellipsoidal cerebral ganglion located between the gut and the cephalic intestine and invested by a fibrillar collagen-like capsule 0.3 μm thick; (b) anterior extracapsular ganglion cell clusters; (c) a peripheral nerve plexus locally thickened at the level of the epithelial sensory and glandular areas, with extensive synaptic connections. At least two neuron types can be identified within the ganglion: (a) an inner layer close to the central neuropile of the 1st type of neurons, showing a vesicular cytoplasm rich in RER and Golgi complexes processing both round, clear, 25–45 nm in diameter, and dense cored vesicles, 50–80 nm in diameter; (b) an outer layer of the 2nd type of neurons, adjoining the capsule and filled with uniformly dense vesicles, 60–90 nm in diameter. Synaptic endings in the neuropile are provided with clear vesicles and dense cored vesicles or uniformly dense vesicles. The presynaptic side has paramembranous projections channelling the vesicles to the active zone; omega-like profiles are also observed. Thin banded muscle fibres run within the brain. A comparison is drawn with the other turbellarian neuron types described in the literature, to suggest their possible function. The functional implications of the synaptic ultrastructure are discussed.     

Ultrastructure of neuro‐spirocyte synapses in the sea anemone Aiptasia pallida (Cnidaria,Anthozoa, Zoantharia)

Using transmission electron microscopy of serially sectioned tentacles from the sea anemone Aiptasia pallida, we located and characterized two types of neuro‐spirocyte synapses. Clear vesicles were observed at 10 synapses and dense‐cored vesicles at five synapses. The diameters of vesicles at each neuro‐spirocyte synapse were averaged; clear vesicles ranged from 49–89 nm in diameter, whereas the dense‐cored vesicles ranged from 97–120 nm in diameter. One sequential pair of synapses included a neuro‐spirocyte synapse with clear vesicles (81 nm) and a neuro‐neuronal synapse with dense‐cored vesicles (168 nm). A second synapse on the same cell had dense‐cored vesicles (103 nm). An Antho‐RFamide‐labeled ganglion cell and three different neurites were observed adjacent to spirocytes, but no neuro‐spirocyte synapses were present. Many of the spirocytes also were immunoreactive to Antho‐RFamide. The presence of sequential neuro‐neuro‐spirocyte synapses suggests that synaptic modulation may be involved in the neural control of spirocyst discharge. The occurrence of either dense‐cored or clear vesicles at neuro‐spirocyte synapses suggests that at least two types of neurotransmitter substances control the discharge of spirocysts in sea anemones. J. Morphol. 241:165–173, 1999. © 1999 Wiley‐Liss, Inc.     

An ultrastructural study of the polyp and strobila of Atorella japonica (Cnidaria,Coronatae) with respect to muscles and nerves

Atorella japonica were observed by TEM to examine the nerve plexus in the capitulum of the polyp and the cross-striated muscle cells of the strobila. The nerve plexus included a number of neuromuscular junctions and many interneural synapses. Neuromuscular junctions contained two types of synaptic vesicle: clear and small (ca 75 nm diam.), and dense cored and large (ca 120 nm diam.). The first type of vesicle always appeared near the presynaptic membrane and the second type was distributed behind the former. In interneural synapses, two types of vesicle which were similar to neuromuscular synaptic vesicles were recognized. They were distributed in a pattern similar to that of the neuromuscular synaptic vesicles, but these vesicles were found on both sides of the two synaptic membranes.     

Dense cored vesicles in presynaptic profiles of the rabbit dorsal lateral geniculate nucleus

We are carrying out a study about the synaptic relations between identified synaptic profiles in the dorsal lateral geniculate nucleus (dLGN) of the rabbit. Here, the types of synaptic vesicle containing profiles of the dLGN are described. There are presynaptic large profiles containing round vesicles and pale mitochondria (RLP terminals) and small profiles that contain round vesicles and dark mitochondria (RSD terminals) which respectively arise from the retina and the visual cortex. Another type of presynaptic profile contains elliptical vesicles (F-boutons) which can be subdivided according to their cytoplasmic content. These F-boutons arise from dLGN interneurons. We have found different sized vesicles that have a dense core within RLP, and F terminals and a possible RSD terminal. The significance of the coexistance of pale and dense cored vesicles in the presynaptic profiles of the rabbit dLGN is discussed.     

Morphological evidence for a direct innervation of the mouse vomeronasal glands

Summary The morphological evidence for a direct autonomic innervation of the mouse vomeronasal glands is presented. Axonal varicosities containing a few densecore vesicles and numerous clear vesicles (36–60 nm in diameter) make synaptic contacts with the secretory cells at the base of the glandular acini. The axonal presynaptic membrane is associated with a distinct dense material and it is separated from the secretory cell by a synaptic cleft of about 12–14 nm. At the postsynaptical level, coated vesicles can be found. Additional postsynaptical specializations have not been observed.     

Acetylcholinesterase activity and type C synapses in the hypoglossal, facial and spinal-cord motor nuclei of rats. An electron-microscope study

Using the electron-microscope technique of Lewis and Shute, we studied the localization of the acetylcholinesterase (AChE) activity in the hypoglossal, facial and spinal-cord motor nuclei of rats. The technique used selectively detects synapses with subsynaptic cisterns (type C synapses) as well as heavy deposits of reaction products in the rough endoplasmic reticulum, in fragments of the nuclear envelope, in some Golgi zones and on parts of the pericaryal plasma membrane, the axolemma and the dendritic membrane. In C synapses, AChE activity was located in the synaptic cleft and on the membrane of presynaptic boutons. Some C synapses exhibited distinct synaptic specialization in the form of multiple 'active zones'. These zones were characterized by dense presynaptic projections, short dilations of the synaptic cleft, and postsynaptic densities localized between the postsynaptic membrane and the outer membrane of the subsynaptic cistern. Within the postsynaptic densities, rows of rod- or channel-like structures were observed. The subsynaptic cisterns were continuous with the positive rough endoplasmic reticulum. The results are discussed in terms of the possible role of C synapses in the regulation of AChE synthesis in postsynaptic cholinergic neurons and/or in the regulation of AChE release into the extracellular space as well as in the establishment of new synaptic contacts.     

Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae

The innervation of ventral longitudinal abdominal muscles (muscles 6, 7, 12, and 13) of third-instar Drosophila larvae was investigated with Nomarski, confocal, and electron microscopy to define the ultrastructural features of synapse-bearing terminals. As shown by previous workers, muscles 6 and 7 receive in most abdominal segments “Type I” endings, which are restricted in distribution and possess relatively prominent periodic terminal enlargements (“boutons”); whereas muscles 12 and 13 have in addition “Type II” terminals, which are more widely distributed and have smaller “boutons.” Serial sectioning of the Type I innervation of muscles 6 and 7 showed that two axons with distinctive endings contribute to it. One axon (termed Axon 1) has somewhat larger boutons, containing numerous synapses and presynaptic dense bodies (putative active zones for transmitter release). This axon also has more numerous intraterminal mitochondria, and a profuse subsynaptic reticulum around or under the synaptic boutons. The second axon (Axon 2) provides somewhat smaller boutons, with fewer synapses and dense bodies per bouton, fewer intraterminal mitochondria, and less-developed subsynaptic reticulum. Both axons contain clear synaptic vesicles, with occasional large dense vesicles. Approximately 800 synapses are provided by Axon 1 to muscles 6 and 7, and approximately 250 synapses are provided by Axon 2. In muscles 12 and 13, endings with predominantly clear synaptic vesicles, generally similar to the Type I endings of muscles 6 and 7, were found, along with another type of ending containing predominantly dense-cored vesicles, with small clusters of clear synaptic vesicles. This second type of ending was found most frequently in muscle 12, and probably corresponds to a subset of the “Type II” endings seen in the light microscope. Type I endings are thought to generate the ?fast’? and ?slow’? junctional potentials seen in electrophysiological recordings, whereas the physiological actions of Type II endings are presently not known. © 1993 John Wiley & Sons, Inc.     

Localization of catecholamines and acetylcholinesterase in the terminal nerve fibres of the rabbit myometrium

Summary The autonomic nerves of the myometrium of the rabbit were studied in order to demonstrate simultaneously the adrenergic nature of an axon and the localization of acetylcholinesterase (AChE) in the same axons. The synaptic vesicles of the adrenergic axons and nerve terminals remained partially filled with the electron dense material typical for them after formaldehyde fixation and short incubation time for AChE. AChE stain was localized regularly on the axons which contained agranular synaptic vesicles and also on axons which contained dense cored synaptic vesicles beeing probably adrenergic. The role of AChE on the adrenergic axons is discussed.     

Electron microscopic analysis of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studied by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80-120 nm dense core granules and 30-50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine-beta-hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial parvocellular subnucleus of PVN. Labeled terminal boutons contained 70-100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN. Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN. In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70-120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons. These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.     

Electron-microscopic studies on developing intramural ganglia of the small intestine in human and rabbit fetuses.

Electron-microscopic studies were made on the appearance of synapses in the intramural ganglion (Auerbach) and findings were correlated with the onset and development of intestinal peristalsis in 6- to 30-week-old human and rabbit fetuses from the 12th day after conception until birth. At stage I, in which the small intestine shows no indication of a muscle layer or spontaneous peristalsis, primitive synapses containing several clear vesicles and a few cored vesicles are seen on neuroblasts and their processes (dendrites). At stage II, in which the circular muscle is developed and bidirectional peristalsis occurs, synaptic profiles can be classified into 3 types. Type 1 is the most numerous but seldom shows membrane specificity on the synaptic portion. Types 2 and 3 have small flattened vesicles and small round vesicles, respectively. They are further characterized by thickening of snyaptic membranes and aggregation of small clear vesicles associated with the presynaptic membrane. At stage III, the longitudinal muscle layer develops in the small intestine. At this stage, nerve terminals containing mainly cored vesicles have been observed and classified into types 4 and 5, according to their morphology. At stage IV, antiperistalsis no longer occurs and type 6 nerve terminals in the intramural ganglia can be recognized by their densely packed, large-cored vesicles. The possible physiological significance of the nerve terminals has been discussed.     

Ultrastructural features of neuromuscular junctions in the stapedius muscle of Gallus gallus

The ultrastructure of neuromuscular junctions in the twitch fibers of the stapedius muscle of Gallus gallus (domesticus) was investigated as part of a series of neurophysiological studies. Among the morphological features observed were elongated end-plates with numerous large and clear synaptic vesicles mixed with larger dense core vesicles and irregular or aperiodic “active sites” in the presynaptic membrane where synaptic vesicles were focused. The most remarkable features of these junctions were large synaptic clefts (50-80 nm) and the absence of junctional folds in the sarcolemmal surface. Unlike the large periodic junctional folds seen in the neuromuscular junctions of frogs and in the fast twitch fibers of the mammalian stapedius, the preparations studied only show small aperiodic invaginations (primitive folds) in the postsynaptic membranes. This morphological feature remains essentially constant from newly hatched to adult chickens. While these smooth junctions are consistent with earlier findings of inconspicuous junctional folds in the twitch fibers of the chicken posterior latissimus dorsi they are unlike those seen in the fast twitch fibers of the mammalian stapedius muscle, or other twitch fibers in general. The morphological findings of the present study may also suggest that the simple, unmodified neuromuscular junctions in the stapedius of Gallus may be a useful preparation for studies of synaptic membrane structures that employ the freeze-fracture technique.     

The fine structure of the central synaptic contacts on an identified crustacean motoneuron

Summary Small nerve terminals in the neuropile of the brain of the crab Scylla serrata make close contact with the secondary, tertiary and higher order central branches of the reflex eye-withdrawal motoneurons. Most contacts have the characteristics of chemically transmitting synapses in that the presynaptic terminals contain agranular vesicles of 25 to 50 nm in diameter and are separated from the motoneuron by a synaptic cleft of about 16 nm. Some terminals contain synaptic ribbons, others contain a mixture of larger (50 to 80 nm) agranular and also dense cored vesicles. In addition large blunt-ended contacts unaccompanied by vesicles, occur between neurons in the neuropile and the motoneuron. It is suggested that the absence of synaptic contacts over the large primary branches of the motoneuron could explain previous physiological findings that little or no resistance changes can be detected in this part of the neuron during excitation or inhibition.We thank Mrs. Joan Goodrum for the preparation of Fig. 1.     

The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1

We have examined the cytoskeletal architecture and its relationship with synaptic vesicles in synapses by quick-freeze deep-etch electron microscopy (QF.DE). The main cytoskeletal elements in the presynaptic terminals (neuromuscular junction, electric organ, and cerebellar cortex) were actin filaments and microtubules. The actin filaments formed a network and frequently were associated closely with the presynaptic plasma membranes and active zones. Short, linking strands approximately 30 nm long were found between actin and synaptic vesicles, between microtubules and synaptic vesicles. Fine strands (30-60 nm) were also found between synaptic vesicles. Frequently spherical structures existed in the middle of the strands between synaptic vesicles. Another kind of strand (approximately 100 nm long, thinner than the actin filaments) between synaptic vesicles and plasma membranes was also observed. We have examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in vitro using the low angle rotary shadowing technique and QF.DE. The synapsin 1, approximately 47 nm long, was composed of a head (approximately 14 nm diam) and a tail (approximately 33 nm long), having a tadpole-like appearance. The high resolution provided by QF.DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles, forming approximately 30-nm short strands. The head was on the actin and the tail was attached to the synaptic vesicle or actin filament. Microtubules were also cross-linked by a single synapsin 1, which also connected a microtubule to synaptic vesicles, forming approximately 30 nm strands. The spherical head was on the microtubules and the tail was attached to the synaptic vesicles or to microtubules. Synaptic vesicles incubated with synapsin 1 were linked with each other via fine short fibrils and frequently we identified spherical structures from which two or three fibril radiated and cross-linked synaptic vesicles. We have examined the localization of synapsin 1 using ultracryomicrotomy and colloidal gold-immunocytochemistry of anti-synapsin 1 IgG. Synapsin 1 was exclusively localized in the regions occupied by synaptic vesicles. Statistical analyses indicated that synapsin 1 is located mostly at least approximately 30 nm away from the presynaptic membrane. These data derived via three different approaches suggest that synapsin 1 could be a main element of short linkages between actin filaments and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles in the nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural studies on the afferent synaptic input to oxytocin-containing neurons in the paraventricular nucleus of the hypothalamus

A diverse afferent synaptic input to immunostained oxytocin magnocellular neurons of the paraventricular nucleus of the rat hypothalamus is described. By electron microscopy, immunoreactive material is present within cell bodies and neuronal processes and it is associated primarily with neurosecretory granules and granular endoplasmic reticulum. Afferent axon terminals synapse on perikarya, dendritic processes, and possibly axonal processes of oxytocin-containing neurons. The presynaptic elements of the synaptic complexes contain clear spherical vesicles, a mixture of clear spherical and ellipsoidal vesicles, or a mixture of clear and dense-centered vesicles. The postsynaptic membranes of oxytocinergic cells frequently show a prominent coating of dense material on the cytoplasmic face which gives the synaptic complex a marked asymmetry.     

Ultrastructure of calcitonin gene-related peptide-immunoreactive nerve fibres in guinea-pig peribronchial ganglia.

Calcitonin gene-related peptide-immunoreactive (CGRP-IR) nerves within guinea-pig peribronchial ganglia were studied at ultrastructural level using pre-embedding immunohistochemistry. Preterminal CGRP-IR axons were unmyelinated and contained singular immunoreactive dense core vesicles. CGRP-IR axon terminals were filled with numerous non-reactive small clear vesicles and few immunoreactive dense core vesicles. Some of these terminals were presynaptic to large neuronal processes emerging from local ganglion cells. Another population of presynaptic varicosities lack CGRP-IR. Within CGRP-IR terminals, non-reactive clear vesicles were clustered at the presynaptic membrane whereas CGRP-IR large vesicles remained in some distance from the synaptic cleft. The present observations indicate that: (1) at least two neurochemically different types of synaptic input exist to guinea-pig peribronchial ganglia. (2) CGRP-IR presynaptic terminals probably utilize a non-peptide transmitter for fast synaptic transmission, whilst the peptides are likely to be released parasynaptically and may act in a modulatory fashion.     

Interneuronal synapses in the local ganglia of the cat urinary bladder.

The interneuronal synapses of the urinary bladder in the cat were studied by electron microscopy. The great majority of the fibres containing vesicles are found within the ganglia occurring in the trigonum area. Morphologically differentiated synaptic contacts could be observed on the surface of the local neurons and between the different nerve processes. The presynaptic terminals can be divided into three types based on a combination of synaptic vesicles. Type I terminals, presumably cholinergic synaptic terminals, contain only small clear vesicles of 40-50 nm in diameter. Type II terminals, presumably adrenergic terminals, are characterized by small granulated vesicles of 40-60 nm in diameter. Type III terminals, probably of local origin, contain a variable number of large granulated vesicles of 80-140 nm in diameter. Occasionally, a single nerve fibre contacted several (two or four) other nerve processes forming a typical synapse. In other cases, on one nerve cell soma or on other nerve processes there are two or three different-type nerve terminals establishing synapses. It might be inferred from these observations that convergence and divergence can occur in the local ganglia and that cholinergic and adrenergic synaptic terminals can modulate the ganglionic activity. However, a local circuit also can play an important role in coordinating the function of the bladder.