The effects of different methods of fixation on central nervous system synaptic pinocytotic vesicles

Summary Synaptic pinocytotic vesicles (invaginating from the surface membrane) and coated vesicles inside rat mossy fiber endings were counted after the use of different kinds of fixatives. Significantly greater numbers of pinocytotic vesicles and coated pinocytotic vesicles per unit length of membrane were found when osmium was used as the first fixative. A high positive correlation was found between these values and the number of coated vesicles per unit area of mossy fiber ending profiles. These results emphasize the need for caution when considering the theory that in vivo synaptic vesicle recycling involves a coated vesicle invagination of the surface membrane followed by internalisation and loss of coat of the vesicle.The authors are indebted to Mrs. M.L. Brito and M.M. Pacheco and Mr. L.B. Nunes for technical assistance. This work has been supported by I.A.C. (Lisbon)     

Fine structure of synapses in the hippocampus of the rabbit with special reference to dark presynaptic endings

Summary The stratum radiatum of h ₃ and h ₄ in the hippocampus of the rahbit, where the mossy fiber endings are distributed, was investigated under the electron microscope. These regions contain a certain number of electron dense presynaptic endings. These are characterized by highly dense synaptic vesicles and mitochondrial matrices. The dense endings are not considered as degenerated. Electron dense silver particles, substituted for zinc, occurred on the synaptic vesicles of these dense terminals as well as the mossy fiber endings after the application of Timm's histochemical method modified for electron microscopy. It is concluded that the dark synaptic endings observed might represent mossy fiber terminals in a special functional phase, or might be the result of structural alteration in the course of tissue preparation. The zinc localized in the synaptic vesicles is thought to be associated with the neurotransmitter present in these endings.     

Ultrastructure of synaptosomes from one-day old and adult rat brain stem

Summary The ultrastructure and protein content of the five subfractions of the crude mitochondrial fraction from the brain stem of the 1-day old and adult rat was examined. The morphological composition of the subfractions after fixation in glutaraldehyde and osmiumtetroxide in the adult rat brain stem resembled that previously reported for the whole brain; synaptosomes sedimented in a sucrose gradient in subfractions C and D. In the 1-day old rat, mature synaptosomes were found in subfractions A, B, C and D; E contained mainly free mitochondria. 80–95% of the processes in the adult and 10–30% in the 1-day old rat contained synaptic vesicles which were of four types: (1) small agranular vesicles (2) large dense core vesicles (3) large agranular vesicles (4) coated vesicles. Pre- and postsynaptic membrane thickenings were demonstrated in many nerve-ending particles. In the subfractions of the 1-day old rat the protein content was one half and the distribution resembled that in the adult. Evidently nerve endings develop faster in the brain stem than in cortical areas; a serotoninor adrenergic origin of the early synaptosomes is suggested.This study was supported by a grant from the Paulo Foundation.     

Cytomorphological alterations of mossy fiber boutons of the hippocampus produced by 3-acetylpyridine,methoxypyridoxine, reserpine,and iproniazid

Summary The hippocampal mossy fiber boutons of the rabbit were studied with phase and electron microscopy. The injection of 3-acetylpyridine, methoxypyridoxine, and reserpine diminishes the conspicuous osmiophilic density of the mossy fiber boutons in comparison to similar regions from nontreated animals as observable in phase microscopy. However, electron micrographs of the same samples show little or no diminution in the number of those synaptic vesicles consisting of a clear homogeneous center (Type I). Treatment with monoamine liberator, reserpine, results in the same cytomorphological appearance of the boutons as with convulsant agents. The number of synaptic dense-core vesicles (Type II) is not altered after treatment with the convulsant agents or reserpine.A certain extra-vesicular substance and a certain granular component of the vesicular membranes of Type I vesicles is progressively reduced after treatment with all of these drugs. It is suggested that this accounts for the decreased density by phase microscopy.The monoamine oxidase inhibitor, iproniazid, increases the density of the extra-vesicular substance as well as the particles attached to the vesicular membranes of Type I vesicles.It is suggested that these osmiophilic particles contain the biogenic monoamines (in this instance probably serotonin and/or histamine) and that in acute experiments the liberation of these neurotransmitters is not related to a disappearence of dense-core vesicles concommitant with a depletion of neurotransmitters but is from particles in the extra-vesicular substance and the granular component of the vesicular of the Type I vesicles.Furthermore, the functional role of zinc in the synaptic vesicles of mossy fiber boutons of the hippocampus is discussed in regard to a possible storage mechanism for biogenic monoamines.This study was partly supported by USPHS Grant 5 P10 ESOO159.     

Isolation of small agranular synaptic vesicles of rat brain by gel filtration chromatography

I attempted to isolate synaptic vesicles by gel filtration. The rat brain synaptic vesicles in a synaptosomal lysate were collected by ammonium sulfate salting-out and fractionated on a Sephacryl S-500 with a mean exclusion size of 200 nm. Peak I at the void volume contained large vesicular membranes and coated vesicles besides synaptic vesicles; Peak II consisted almost entirely of small agranular synaptic vesicles of 40-50 nm diameter; and Peak III comprised soluble proteins. Western blotting revealed that components of 72 kDa in peaks I and II reacted with an anti-H(+)-ATPase A-subunit antibody [Moriyama et al. (1995) FEBS Lett. 367, 233-236]. When examined for Mg(2+)-ATPase activity, peak I showed specific activity of 4.52 ( micromol ATP hydrolyzed/mg protein/30 min), while that of peak II was as low as 0.22. As estimated from the inhibition by bafilomycin A(1) [Bowman et al. (1988) PROC: Natl. Acad. Sci. USA 85, 7972-7976], the percentage of H(+)-ATPase as to total Mg(2+)-ATPase, 18-22%, was unchanged, indicating no accumulation of the H(+)-ATPase in peak II even on the chromatography. In brief, the small agranular synaptic vesicles in peak II showed little or no Mg(2+)-ATPase activity, although they reacted with the H(+)-ATPase antibody. The reason for this is obscure. Mg(2+)-ATPase might not be a constituent of small agranular synaptic vesicles of rat brain.     

Synaptic ribbons during postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus)

Summary Synaptic ribbons, functionally enigmatic structures of mammalian pinealocytes, were studied during the postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus). On day 4 post partum, synaptic ribbons appear in cells that have already started to differentiate into pinealocytes. Between days 4 and 9, an increase in the number of synaptic ribbons occurs, concomitant with the continuing differentiation of the pineal tissue. Between days 9 and 16, when differentiation of this tissue is almost completed, the number of synaptic ribbons decreases and approaches that characteristic of the adult pineal gland. During development, the synaptic ribbons increase in length, and dense core vesicles are frequently found in the vicinity of these structures. It is assumed that a functional relationship exists between dense core vesicles and the synaptic ribbons, which are considered to be engaged in a certain form of secretory activity of the mammalian pineal gland.Supported by the Deutsche Forschungsgemeinschaft     

Effect of the zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) on hippocampal mossy fiber calcium signals and on synaptic transmission

An important pool of chelatable zinc is present in the synaptic vesicles of mossy fiber terminals from hippocampal CA3 area, being zinc released following single or repetitive electrical stimulation. Previous studies have suggested different synaptic roles for released mossy fiber zinc, including the inhibition of presynaptic calcium and of postsynaptic N-methyl-D-aspartate (NMDA) and gamma amino-butyric acid (GABAA) receptors. The effect of endogenously released zinc on mossy fiber long-term potentiation (LTP) induction also is not yet established. We have investigated the effect of the permeant zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) on mossy fiber calcium and on synaptic transmission, before and during the application of LTP-inducing stimulation. We have found, using the calcium indicator Fura-2, that single and tetanically-evoked mossy fiber calcium signals are both enhanced in the presence of 20 microM TPEN, while the single field potentials are unaffected. As expected, no effect was observed on the single calcium signals or field potentials obtained at the CA3-CA1 synapses, from the CA1 area, which has a lower concentration of vesicular zinc. These results support the idea that at the hippocampal mossy fiber synapses, released zinc inhibits presynaptic calcium mechanisms. A higher concentration of TPEN (100 microM) significantly reduced mossy fiber synaptic transmission but did not prevent the induction of mossy fiber LTP, suggesting that zinc is not required for the formation of this form of LTP.     

Electron microscopic histochemistry of acetylcholinesterase of rat brain by Karnovsky's method

Summary Karnovsky's electron microscopic acetylcholinesterase method was successfully applied to rat brain fixed by vascular perfusion with either 2% glutataldehyde or 4% formaldehyde. 2% glutaraldehyde showed better fine structure but worse preservation of the enzyme than 4% formaldehyde.In the neuropil of the caudate nucleus, locus coeruleus and dorsal nucleus of the vagus, AChE activity was most intensely demonstrated on the plasma membranes of preterminal axons and somewhat less strongly on those of axon terminals and contacting dendritic branches. The axoplasm and synaptic vesicles were usually negative, while the cytoplasm and neurotubules of the dendritic branches showed some activity. In the nodule and uvula of the cerebellum moderate activity was exhibited on the synaptic contacts between the mossy fiber endings and granule cell dendrites. In the hypothalamus and other autonomic regions the characteristic coexistence of AChE and granulated vesicles of axon terminals could be demonstrated.In the perikaryon of positive nerve cells, AChE was observed strongly in the cytoplasm, disseminated irregularly or attached to the endoplasmic reticulum, while it was absent in the mitochondria and lysosomal dense bodies.     

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.     

Thyroidectomy induces coated pit formation on cerebellar mossy fiber terminals

Summary The influence that thyroid hormones have on the development of the cerebellar cortex is well known. Their absence in adults leads to important functional changes probably related to abnormalities in nerve terminal activity, although no morphological alterations have hitherto been described. Using ultrastructural morphometric methods, we have studied the effects of thyroidectomy on the cerebellar cortex mossy fiber terminals of adult rats with different survival periods. No significant changes in the volume and surface area of these terminals were found. The numbers of synaptic vesicles in groups 7, 15 and 30 days after thyroidectomy were significantly greater than in controls. A significant increase in coated pit formation was found when thyroidectomized groups were compared with controls. This increase is due to the lack of thyroxin since it can be counteracted by thyroxin administration. Whether this increased coated pit formation is a membrane retrieval mechanism or is related to the uptake of extracellular molecules remains to be determined.     

Synapsin I deficiency results in the structural change in the presynaptic terminals in the murine nervous system

Synapsin I is one of the major synaptic vesicle-associated proteins. Previous experiments implicated its crucial role in synaptogenesis and transmitter release. To better define the role of synapsin I in vivo, we used gene targeting to disrupt the murine synapsin I gene. Mutant mice lacking synapsin I appeared to develop normally and did not have gross anatomical abnormalities. However, when we examined the presynaptic structure of the hippocampal CA3 field in detail, we found that the sizes of mossy fiber giant terminals were significantly smaller, the number of synaptic vesicles became reduced, and the presynaptic structures altered, although the mossy fiber long-term potentiation remained intact. These results suggest significant contribution of synapsin I to the formation and maintenance of the presynaptic structure.     

Fine structure of nervous elements in the pancreas of some vertebrates

Summary The innervations of the exocrine and endocrine pancreas of some vertebrate animals were studied by electron microscopy. The pancreas of the bat and monkey contained ganglion cells in the interlobular connective tissue or between acinar cells. Unmyelinated nerve fibers ran through the interlobular connective tissue and reached the exocrine and endocrine parts, and terminated there as the endings. The nerve endings within the pancreas could be divided into four types: 1. Type 1-a of the nerve ending contained only agranular synaptic vesicles of about 500 Å in diameter. 2. Type 1-b characterized by containing agranular synaptic vesicles and some large cored vesicles (1,000 Å diameter). These two types of nerve endings might belong to the cholinergic (parasympathetic) endings. 3. Type 2-a contained small cored vesicles and agranular synaptic vesicles along with a few large cored vesicles. 4. Type 2-b was characterized by containing vesicles of the same size as those of agranular synaptic vesicles, and a majority of these vesicles contained bar-shaped crystalloids. This ending also contained a few large cored vesicles. These nerve endings of Type 2-a and 2-b might be the adrenergic (sympathetic) endings.     

Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGlu_u that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength.

This study uses several high-resolution imaging techniques to investigate the structural correlates of presynaptic potentiation at hippocampal mossy fiber boutons, observing an increase in release sites and in release synchronicity accompanied by synaptic vesicle dispersion in the terminal and accumulation at release sites, but no modulation of the distance between calcium channel and release sites.     

Ultrastructural changes in granule cell somata and mossy fibers of the rat hippocampus during picrotoxin-induced convulsions

Summary Ultrastructural changes in hippocampal granule cells, mossy fibers and mossy fiber boutons were examined following the administration of picrotoxin in adult rats. Generalized seizures occurred within 5–10 min after the intraperitoneal injection of picrotoxin. The electron-microscopic examination of hippocampal tissues from rats that had been perfused with fixative during the seizure revealed that the large dense-core vesicles increased in number and accumulated on the presynaptic membranes of mossy fiber boutons; some of these vesicles appeared to be fused with the membranes, and omega-shaped exocytotic profiles were frequently seen. Furthermore, greatly increased numbers of coated vesicles (60–90 nm in diameter) were observed on the maturing faces of Golgi fields of granule cells. Thus, our study not only indicates an increased incidence of exocytosis of large dense-core vesicles during picrotoxin-induced seizures, but also suggests that these vesicles are replaced in excess from the perikaryon of the granule cell.     

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.     

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.     

Immunocytochemical localization of coated vesicle protein in rodent nervous system

Immunocytochemistry has been used to study the distribution of the major 180,000-mol wt protein of coated vesicles in rodent cerebellum. An antibody to the coat protein was prepared in rabbits and characterized by immunodiffusion and immunofixation of polyacrylamide gels. At the light microscope level the protein was primarily localized in punctate profiles surrounding Purkinje cells and within the cerebellar glomeruli. At the electron microscope level the punctate distribution was confined to presynaptic terminals of basket and Golgi II neurons as well as mossy fiber terminals of the glomeruli. This label was heaviest on the lattice coat of coated vesicles but, in addition, label was found within the presynaptic axoplasm and along the cytoplasmic surface of the plasmalemma. Coated vesicles in cell somata were labeled as well as the cytosol around groupings of these vesicles. These data suggest that there may be two forms (or more) of coated vesicle protein in neurons, a lattice form associated with coated vesicles and a soluble form associated with the cytoplasmic matrix.     

Light and electron microscopic immunocytochemical localization of clathrin in rat cerebellum and kidney

The precise cellular and subcellular locations of coated vesicle protein, clathrin, in rat kidney and cerebellum have been visualized by immunocytochemical techniques. In the renal tubular epithelia, clathrin-positive products were found on both free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No clathrin was observed in the cisternae of RER or the Golgi apparatus. Clathrin-positive reaction products could also be seen on coated pits, coated vesicles, Golgi-associated vesicles, basolateral cell membrane, the ground substance, and in the autophagic vacuoles. In cerebellar Purkinje and granule cell bodies, reaction products were seen localized on coated vesicles, on the budding areas from the Golgi-associated membrane and Golgi-associated vesicles. Furthermore, the membrane of the multivesicular body, the bound-ribosomes, and the ground substance were also stained. In the myelinated axon, the clathrin appeared to be concentrated on certain segments and seemed to fill in the space between neurotubules and some vesicles. In certain synaptic terminals clathrin was often seen attached to presynaptic vesicles, presynaptic membrane, and post-synaptic membrane. However, in most mossy fibers, some synaptic vesicles were not stained. These observations suggest that clathrin is synthesized on bound and free ribosomes and discharged into the cytosol where it becomes associated with a variety of ground substances and assembles on coated pits, coated vesicles, Golgi-associated vesicles, presynaptic vesicles, and pre- and postsynaptic membranes. Clathrin may be finally degraded in autophagic vacuoles.     

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.