Zinc Iodide-osmium Research Articles

Zinc iodide-osmium tetroxide reaction: its effect on the synaptic vesicles in locust neuromuscular junctions and its chemical basis

The zinc iodide-osmium tetroxide reaction (ZIO) was first used in neurobiological research by Maillet (Bull. Ass. Anat. 53, 233; 1968). Subsequently several authors have shown that, under appropriate conditions, ZIO stains mainly the interior of synaptic vesicles. The substrate of this reaction is under discussion, since ZIO can also react with other subcellular structures in a variety of tissues, e. g. mitochondria, endoplasmic reticulum, dictyosomes and lysosomes. Additionally, in vitro substances as different as some aminoacids, catecholamines, aldehydes and phospholipids (Pellegrino de Iraldi, Experientia 33, 1; 1977) can yield black precipitations with ZIO.Our studies were done with the motor nerve terminals at the femoral retractor unguis muscle of the locust (Locusta migratoria). These terminals are chiefly the endings of excitatory motoraxons and are characterized by the presence of electron lucent vesicles and by an accumulation of mitochondria.

Membranes in lupin root nodules. I. The role of Golgi bodies in the biogenesis of infection threads and peribacteroid membranes.

The process of infection of lupin nodule cells by rhizobia was examined using thin-section and freeze-fracture electron-microscopic techniques to characterize the properties of different membranes and to establish relationships between them. The membranes of the Golgi bodies and the endoplasmic reticulum stained with zinc iodide-osmium tetroxide but not with phosphotungstic acid or silver. By contrast the infection thread membranes, peribacteroid membranes, plasma membranes and membranes of cytoplasmic vesicles did not stain with zinc iodide-osmium tetroxide but stained with phosphotungstic acid and silver. The peribacteroid membranes and plasma membranes are, however, different from each other since the particle density on the E face of freeze-fracture replicas of plasma membranes was twice that on the E face of the peribacteroid membranes. An examination of the tips of the infection threads in the cytoplasm of the plant cells, showed that the rhizobia bud off from the infection threads enclosed in the infection thread membranes. The rhizobia continue to divide still surrounded by membranes of plant origin, namely the peribacteroid membranes. Cytoplasmic vesicles are observed in both thin-section and freeze-fracture preparations of nodule tissue closely associated with, and apparently produced by, Golgi bodies. Formation of the walls and membranes of the infection threads and of the peribacteroid membranes involves fusion of the cytoplasmic vesicles with these membranes. It is proposed that the process of infection of plant cells in lupin nodules involves a change in the function of the Golgi body system for the biogenesis of plant cell walls and plasma membranes to include the synthesis of the walls and membranes of the infection threads and also the peribacteroid membranes.

Studies on the fine structural localization of zinc iodide-osmium reaction in the brain. III. Some characteristics of localization in the synaptosomes

Synaptosomes from rat cerebral cortex were impregnated in the zinc iodide--osmium (ZIO) solution. The fine structural localization of the ZIO impregnation product was studied and, in addition, the function-dependent features of the reaction were examined after electrical stimulation or potassium chloride treatment. It was revealed that: (i) Aldehyde prefixation resulted in an increase in the number of reactive synaptic vesicles in all types of synaptosomes; (ii) Electrical stimulation decreased the number of reactive vesicles in a voltage dependent manner; (iii) Potassium chloride treatment also reduced the reactivity of vesicles; the reduction was dependent on the concentration of potassium and duration of treatment; (iv) Experimental interventions leading to the release of neurotransmitters from the synaptic vesicles and to fatigue of the nerve terminals also resulted in a decrease of the ZIO-reaction product of synaptic vesicles in a manner proportional to the strength of stimuli.

An ultrastructural classification of the neuronal cell bodies of the rat dorsal root ganglion using zinc iodide-osmium impregnation.

Zinc iodide-osmium (ZIO) impregnation of rat dorsal root ganglia differentially stained various elements in the neuronal cells, particularly their Golgi bodies. On the basis of this differential ZIO staining dorsal root ganglion neurones have been classified into seven types. The ultrastructure of these is described and the numbers of each type in the L4 dorsal root ganglion have been determined. Prolonged nerve stimulation did not change the relative numbers of the different cell types suggesting that none of the differences between cell types represents differences in their state of activity. The possibility is discussed that differences in morphology may reflect differences in neurotransmitter function.

The reorganization of synaptic connexions in the rat submandibular ganglion during post-natal development.

1. The innervation of neurones in the submandibular ganglion of neonatal and adult rats has been studied with intracellular recording, and light and electron microscopy. 2. Intracellular recordings from neurones in isolated ganglia from adult animals showed that about 75% of the ganglion cells are innervated by a single preganglionic fibre. 3. However multiple steps in the post-synaptic potential (about five on average) were elicited in ganglion cells from neonatal animals by graded stimulation of the preganglionic nerve. The same result was obtained when the preganglionic fibres were stimulated at their emergence from the brainstem, indicating that neonatal neurones are innervated by several different preganglionic nerve cells. 4. The number of preganglionic fibres innervating individual ganglion cells gradually decreased during the first few weeks of life, and by about 5 weeks each ganglion cell was generally contacted by a single preganglionic axon. 5. Synapses were made on short protuberances in the immediate vicinity of the neuronal cell bodies in both neonatal and adult ganglia as shown by staining presynaptic boutons with the zinc-iodide osmium method, injection of horseradish peroxidase into ganglion cells, and electron microscopical examination. 6. Electron microscopical counts of synaptic profiles per ganglion cell perimeter showed that the number of synaptic contacts made on ganglion cells actually increased during the first few post-natal weeks, when the number of axons innervating each neurone was decreasing. 7. These results show that in the rat submandibular ganglion there is a reorganization of neuronal connexions during the first few weeks of life which results in a transition from multiple to generally single innervation of ganglion cells.

Recent findings in oogenesis of Drosophila melanogaster III. Lysosomes and yolk platelets

ABSTRACT The role played by the vitellogenic oocytes of Drosophila melanogaster in relation to the elaboration of material taken from the haemolymph is examined by ultrastructural cytochemistry. As revealed by the Gomori procedure, acid phosphatase occurs widely over the forming yolk platelets of the cortical and central ooplasm. A number of Golgi apparatuses in the cortical ooplasm are also positively stained with lead precipitates. With the proceeding of the ovarian development it becomes progressively more difficult to demonstrate cytochemically the enzyme over the yolk platelets. In stage 9-10 chambers the acid phosphatase is restricted to the so-called associated body, while the rest of the yolk platelet appears devoid of lead deposits. By using a osmium zinc iodide (OZ1) complex as a preferential staining method for the Golgi apparatus, it has been shown that, apart from the apparatus itself, a number of OZI deposits occur over the superficial layer of the forming yolk platelets. When mature yolk plate-lets are formed at later stages, the OZI deposits in the yolk platelets come to be restricted to the cap-like region of the superficial layer which contains the associated body. In vitellogenic oocytes, both the internal lining of the limiting membrane of the forming yolk platelets and the associated body of the mature yolk platelets react positively, to cyto-chemical methods to demonstrate carbohydrates. The present findings are interpreted as indicating the involvement of lysosomal enzymes in the process of maturation of the yolk material. The suggestion is also made that such an involvement is required to accomplish a selective hydrolysis of those blood proteins which have been taken in by vitellogenic oocytes along with yolk precursors.

Affinity of zinc iodide-osmium stain for sensory endings of snake muscle spindles

Affinity of zinc iodide-osmium stain for sensory endings of snake muscle spindles

Ontogeny and Cytochemistry of Alkaloidal Vesicles in Laticifers of Papaver somniferum L. (Papaveraceae)

Development of alkaloidal vesicles in laticifers of opium poppy, Papaver somniferum L., was investigated at the ultrastructural level. Laticifer initials possessed abundant endoplasmic reticulum throughout their dense cytoplasm. During differentiation the endoplasmic reticulum organized into long, folded sheets that were parallel to the longitudinal walls along the periphery of the cell. Vesicles appeared to be derived from dilation of endoplasmic reticulum. This relationship was confirmed through cytochemical data obtained with zinc iodide-osmium tetroxide and osmium tetroxide impregnation. Alkaloidal vesicles had electron-dense regions or caps that occurred early in laticifer differentiation, but these caps became less conspicuous in mature cells. Caps appeared to be derived from small particles which condensed along the inner surface of the vesicle membrane and subsequently accumulated at one or two positions along the membrane of the vesicle.

Morphological changes of the toad adrenal tissue grafted in the median eminence-pars distalis region

The present investigation was designed to determine the morphological changes under-gone by the adrenal gland when grafted into the hypophysial region, paying special attention to the possibility that it might differentiate into pars distalis-like cells. The pars distalis of the hypophysis of 33 male and female toads was carefully dissected out, leaving the vascular supply, and the neural afferents, to the median eminence and neurointermediate lobe intact. Then a fragment of adrenal tissue from a donor toad was implanted in the region that had been occupied by the pars distalis. The operated animals were killed 1, 2, 3, 4, 5, 10 and 12 weeks after the operation. Within each group one animal was used for light microscopic studies and the others for electron microscopy. The graft samples obtained from the 5 week group animals were fixed in glutaraldehyde and then incubated with either amoniacal silver or with zinc iodide-osmium tetroxide mixture at pH 4.4. These two methods stain selectively the noradrenaline storing granules. The grafted adrenal tissue became revascularized by the portal vessels. Histochemical studies did not reveal the presence of pars distalis-like cells in the graft, even 3 months after the operation. Within the first 2 weeks after the operation, a population of cortical cells underwent involution, whereas a second type of cortical cells remained unchanged. During this phase two types of chromaffin cells, similar to those observed in the gland in situ, were distinguished. During the chronic experimental phase all cortical cells showed involution and only one type of chromaffin cell was identified. The latter were loaded with chromaffin granules and displayed numerous processes contacting the perivascular space. The histochemical characteristics of these cells indicate that they are noradrenaline secreting cells. It is concluded that under the present experimental conditions, the grafted adrenal tissue does not differentiate into pars distalis and that only one cell type of the transplanted tissue continues to secrete its specific cell product.

Significance of the maillet method (ZIO) for cytochemical studies of subcellular structures.

The present knowledge on the methodology, specificity, localization, cytochemical significance and experimental variations of the zinc iodide-osmium tetroxide (ZIO) staining are reviewed. Some new findings supporting the concept that ZIO reacts with -SH groups in some structures under specific conditions are also communicated. In this respect the effect of in vitro pretreatment with -SH reagents on ZIO staining, already described in different kinds of synaptic vesicles, was studied in rod photoreceptor outer segments. It was observed that the intra- and extradiskal electron opaque deposits, present in untreated rod outer segments directly stained with ZIO at 4 degrees C for 2 h, were enhanced by previous incubation with dithioerythrytol (DTE), which protects -SH groups and reduce -S-S-bridges; when N-ethyl-maleimide (NEM), a -SH blocking agent was used directly or after DTE, the electron opaque deposits were considerably reduced or abolished. Furthermore the influence of previous incubation with cysteine on ZIO staining was studied in the rat pineal gland. In the controls ZIO made at 4 degrees C for 2 h, reacts strongly with the matrix of the small granulated synaptic vesicles in the pineal nerves; no electron opaque deposits are seen in the membranes of the vacuolar system. Previous incubation with cysteine 5 mM in phosphate buffer makes negative ZIO reaction in the matrix of the synaptic vesicles while with a concentration of 50 mM cysteine, a partial recovery is observed. In both concentrations the membranes of the vacuolar system appeared covered by patchy electron opaque deposits.

The zinc iodide-osmium tetroxide (ZIO) reaction on nerve endings in the median eminence of the rat under normal and experimental conditions

The reaction of nerve endings in the median eminence of the rat to zinc iodide-osmium tetroxide (ZIO) staining was examined electron microscopically under normal and experimental conditions. The experimental condition of catecholamine exhaustion in the nerve endings was induced by the administration of H44/68 and reserpine. Vesicles in the terminals of catecholaminergic nerves reacted similarly to ZIO staining in both normal and experimental material. The majority of synaptic vesicles in various terminals gave a positive ZIO reaction. The neurosecretory elementary granules, however, failed to react with ZIO. On the other hand, some nerve terminals in the external layer of the median eminence showed a strong positive reaction in the cytoplasmic matrix, in mitochondria as well as in synaptic vesicles. These findings strongly suggest that the ZIO-positive substance in nerve terminals is not the transmitter itself, i.e. the monoamine, but rather represents a range of substances commonly found in various kinds of synaptic vesicles and is probably proteinaceous in nature. A brief discussion is also given on the difference in ZIO reactivity between neurosecretory elementary granules and small vesicles in the hypothalamo-hypophyseal tract.

Osmium zinc iodide staining of Golgi elements in oocytes of Triturus cristatus.

Developing oocytes of the newt Triturus cristatus were studied in order to clarify the role played by the Golgi apparatus in the formation of yolk. The cytochemical method used for this purpose was that of Maillet (1968) which employs an Osmium Zinc Iodide (OZI) complex. Previtellogenic oocytes reveal a pattern of OZI staining only after hormonal (HCG) stimulation, following which both the Golgi apparatus and the multivesicular bodies are stained. Vitellogenic oocytes taken from non-hormonally stimulated females reveal OZI deposits in a number of vesicles peripheral to the Golgi apparatus as well as within the superficial layer of the forming yolk platelets. Following hormone stimulation, many of the Golgi apparatus located in the central ooplasm of vitellogenic oocytes have all their cisternae blackened by the OZI deposits; other apparatuses,more peripherally located, remain essentially unchanged in their staining pattern. Further, a large number of OZI stained vesicles becomes visible in the vicinity of the Golgi apparatus and within the superficial layer of the forming yolk platelets. The present findings are interpreted as indicating the occurrence of fusion between Golgi derived vesicles and forming yolk platelets. It is also suggested that the vesicles in question function as carriers of Golgi produced enzymes which are presumably required to accomplish the final elaboration of the yolk material.

Morphological changes induced by -SH reagents in rod photoreceptor outer segments.

Rat retinas were treated in vitro with -SH reagents and "stained" with zinc iodide-osmium tetroxide (ZIO). Dithioerythritol (DTE), an -S-S-reducing agent, increased the electron opaque deposits observed after ZIO staining in the intra- and extradiskal spaces of the rods. N-ethyl-maleimide (nem), an -SH blocking agent, applied directly or after DTE, blocks the ZIO reaction. Furthermore, after treatment with NEM, distorted tubular and vesicular structures are substituted for the stacks of disks. These results strongly suggest that ZIO reacts with -SH groups in rod outer segments. They also indicate that SH-groups play an important role in the structural organization of rod outer segments.

Spatial distribution of synapses onto thoracic motor neurones in locusts.

Aggregates of synaptic vesicles, stained black by the zinc iodideosmium procedure, can be visualised with the light microscope in 1 mum plastic sections. This allows the main branches of a neurone to be reconstructed relatively rapidly and the associated vesicle aggregates to be plotted. By resectioning, the identity of the vesicle aggregates has been confirmed with the electron microscope. Two flight motor neurones in the mesothoracic ganglion of the locust have been examined. One is identified as a dorsal longitudinal muscle motor neurone (muscle 112) and the other is probably a subalar neurone (muscle 99). Both have a large density of vesicle aggregates on the neuropilar segment, the widest part of the main neuronal axis, but few on the neurite within 250 mum of the cell body. The larger branches arising from the neuropilar segment tend to have a lower density of aggregates than fine branches, which suggests that synapses to the branches may occur mainly on the distal twigs. These results are an important preliminary step in determining the integrative functions of such neurons and have immediate implications in the interpretation of microelectrode recordings.

Mechanism for sensillum fluid flow in trichogen and tormogen cells of Phormia regina (Meigen) (Diptera: Calliphoridae)

Electron microscope observations of the tormogen and trichogen cells of the black blow fly ( Phormia regina) chemoreceptor sensillum indicate that an osmium zinc iodide positive (ZIO) substance(s) is conveyed by transport vesicles into the sensillum fluid cavity. Large concentrations of this material(s) were noted between adjacent microvilli which project into the fluid cavity. This particular localization of ZIO-positive substances suggests that they may form part of a fixed hyperosomotic environment which subsequently assists in the flow of the sensillum fluid into the fluid cavity. The formation of ZIO positive material(s) and chemosensory nerve firing are both inhibited by p-chloromercuri benzoate (PCMB, 5 x 10 −4M). Based on the binding affinity of PCMB to sulfhydryl groups, the ZIO-stained substance which is transported to the sensillum fluid cavity may be lipoprotein.

Rôle of axonal transport in maintaining central synaptic connections.

Synaptic boutons on the somata of rat hypoglossal neurones have been stained with Zinc Iodide-Osmium, and counted using light microscopy. Two weeks after Silastic cuffs containing colchicine or vinblastine were applied to the hypoglossal nerve, the number of boutons on parent nerve cell bodies was significantly lower than controls. These drugs inhibited retrograde axonal transport of horseradish peroxidase, but did not affect neuromuscular conduction or the number of axons in the nerve trunk. Cuffs containing lignocaine did not affect boutons or axonal transport significantly.

Ultrastructural and pharmacological studies on the afferent synapse of lateral-line sensory cells of the African clawed toad, Xenopus laevis

Lateral-line sensory cells of Xenopus show considerable similarity to central nervous system synapses in their responses to the zinc iodide-osmium tetroxide, and bismuth-iodide cytochemical staining techniques. Both reserpine and FLA-63 cause a reduction in the diameter and electron density of the synaptic bars, and a dramatic loss of synaptic vesicles surrounding the bars. Aminooxy acetic acid and 3-mercaptopropionic acid cause no significant changes in the sensory-cell afferent synaptic apparatus. The results are discussed in the light of the known effects of the drugs. It is concluded that a monoamine rather than an amino acid may act as the afferent transmitter.

Localizing -SH groups in monoaminergic synaptic vesicles with the mixture of zinc iodide-osmium tetroxide (ZIO)

In monoaminergic granulated vesicles the mixture of zinc iodide-osmium tetroxide (ZIO) stains two compartments: the core and the matrix, the latter being more intensively stained than the core. Rat pineal glands were incubated in0.1 M N-ethyl-maleimide (NEM). It was observed that NEM blocks ZIO reaction both in the matrix and the core, whereas the controls were fully reactive. This finding strongly suggests that ZIO reaction is due to -SH groups and can be correlated with the effect of NEM on uptake processes.

Electron cytochemical demonstration of -SH groups in the synaptic vesicles of photoreceptor cells with the mixture of zinc iodide-osmium tetroxide.

Se demuestra, en un estudio in vitro, que el ditioeritriol (DTE), agente reductor de grupos -S-S-aumenta, la reactividad de las vesículas sináapticas de las células fotorreceptoras de la rata para la mezcla de yoduro de zinc-tetrozido de osmio (ZIO), a 4°C durante 2 h, mientras que la N-etil-maleimida, bloquenante de grupos-SH, bloquea la reacción en los testigos y en los tratados previamente con DTE. Se concluye que ZIO a 4°C durante 2 h revela grupos -Sh en las vesiculas sinápticas estudiadas.

The terminal autonomie nervous system in the pancreas of newts. Electron microscopic investigations using acetylcholinesterase and zinc iodide-osmium tetroxide reactions

The intrinsic innervation in the pancreas of newts Triturus vulgaris, Tr. montandoni and Notophthalmus. All the investigated species display: cholinergic innervation better developed than adrenergic, no qualitative differences regarding the fine structure of nerve fibers and terminals as compared to that previously described in the pancreas or in other glands, axo-axonal contacts. Their functional signification as peripheric synapses is suggested. Frequently neuro-glandular junctions with or without membrane specializations. The occurrence of the latter is discussed in connection with peripheral transmission of nervous impulse.