Timm's Sulphide Silver Method Research Articles

Intravesicular localization of zinc in rat telencephalic boutons. A histochemical study

A transition metal (presumably zinc) has been localized inside the synaptic vesicles of rat telencephalic boutons which make asymmetric synaptic contacts (Gray type I). A modification of Timm's sulphide-silver method for electron microscopy was applied to different areas of the rat forebrain: olfactory bulb, septum, caudate-putamen, amygdaloid complex, neocortex, entorhinal cortex and different parts of the hippocampal formation, viz. subiculum, stratum radiatum and oriens of both regio superior (CA1) and regio inferior hippocampi (CA3), the mossy fibre zone and the hilus fascia dentata and stratum moleculare in the gyrus dentatus. It was observed that the Timm staining in all these areas was located in synaptic boutons. Labelled boutons displayed a population of round clear vesicles and few dense core vesicles and made asymmetric synaptic contacts on dendritic spines. Silver granules in these boutons were confined to only a minor fraction of the round clear vesicles (approximately 10%). Labelled boutons occasionally showed silver granules in the synaptic clefts. The possible involvement of vesicular zinc in synaptic transmission is discussed.

Selenium in the Paneth cells

Two histochemical methods, Timm's sulphide silver method and the selenium method reveal the presence of two pools of metal ions in the Paneth cells. Ultrastructurally one pool is located in the secretion granules, the other in the cytoplasm.

Intraocular grafts of fresh and freeze-stored rat hippocampal tissue: a comparison of survivability and histological and connective organization.

Intraocular grafts of rat hippocampal tissue, grafted either directly from the immature donor brains (fresh) or after storage in liquid nitrogen at -196 degrees C (freeze-stored), were compared with regard to survivability and histological and connective organization. For direct grafting, pieces of hippocampal tissue from rat embryos (embryonic day 19, E21) and newborn rats (PO) were placed in the anterior eye chamber of adult rats immediately after dissection. For grafting after deep-freeze storage, pieces of hippocampal tissue were taken from rat embryos (E16-E21) and newborn rats (PO), frozen at a cooling rate of 1 degrees C/min in CO2 or N2 vapours after addition of the cryoprotective agent dimethylsolfoxide (DMSO), and stored in liquid nitrogen for 1 to 33 days before thawing and intraocular grafting. From 20 to 68 days after grafting, the recipient rats were sacrificed, their eyes sectioned, and the sections stained with thionine for cell bodies, Timm's sulphide silver method for hippocampal fiber systems and terminal fields, and acetylcholinesterase (AChE) for cholinergic fibers and AChE-positive neurons. When examining the 101 grafted eyes (34 grafted with fresh and 67 with freeze-stored tissue) a significantly lower survival rate of the freeze-stored tissue was found (28 vs. 88%). The survivability of the freeze-stored tissue was age-dependent with no survival at donor ages E16 and PO, while tissue from E18-E21 had a 50% survival rate. The grafts of the freeze-stored tissue were also smaller and showed an increased tendency for fragmentation. When evaluating the structure of the grafts, the deep-freeze storage was found primarily to have been harmful to the dentate granule cells and their precursors. The organization of intrinsic fiber connections followed the pattern known from lesion and intracerebral transplant studies. While demonstrating that immature brain tissue can survive deep-freeze storage and subsequent intraocular grafting, the study also indicates that different schemes may have to be used to get optimal survival of different neuronal populations.

The mode of inheritance of a defect in lamination in the hippocampus of BALB/c mice.

In BALB/c mice the lamination of the pyramidal cell layer of area CA3c of the hippocampus is abnormal in that early-generated neurons are superficial and late-generated neurons are deep. To determine the mode of inheritance of this strain difference, the laminar distribution of mossy fibers and hippocampal pyramidal cells was examined using the Timm's sulfide silver method in BALB/c x C57BL/6 F1 and F2 hybrids, in BALB/cByJ and C57BL/6J mice which were fostered to females of the other strain before receiving their first meal, and in the CXB series of recombinant inbred strains (originally derived using BALB/c and C57BL/6 as progenitor strains). The pattern of hippocampal lamination was classified as "BALB/c-like" if pyramidal cells were present below an intrapyramidal mossy fiber layer or as "B6-like" if only an infrapyramidal mossy fiber layer was present. In both male and female CB6F1 and B6CF1 hybrids the distribution of mossy fibers is BALB/c-like. In 7 of 9 F2 hybrids the distribution was BALB/c-like and in the remaining 2 B6-like. In the cross-fostered mice the pattern was always the same as normally raised mice of the same genotype. Of the recombinant inbred strains, 5 (CXBD, CXBG, CXBH, CXBI, and CXBK) had BALB/c-like hippocampal lamination and 2 (CXBE and CXBJ) had B6-like lamination. These results are consistent with inheritance by means of a single autosomal dominant (or semi-dominant) gene. The provisional name "Hippocampal lamination defect" and gene symbol Hld are suggested. The Hld mutation is only the third known neurological mutation in mice which apparently affects neuronal migration, and the fact that it affects only a single subdivision of the hippocampus indicates that Hld may be a useful tool for future studies of the development of the central nervous system and particularly of the cell biology of neuronal migration and neuronal specificity.

Cellular, histochemical and connective organization of the hippocampus and fascia dentata transplanted to different regions of immature and adult rat brains.

The aims of the present study were to examine the survival and the cellular and connective differentiation of intracerebral transplants of fascia dentata and hippocampus. Pieces of immature dentate and hippocampal tissue were taken from late embryonic (E18) and early postnatal (1-9 days old) rats and transplanted into the brains of 1- to 13-day-old and adult rats. After survival times from 4 days to 2 years the cellular and connective organization of the transplants was monitored in parallel series of sections stained with thionin (cell bodies), Timm's sulphide silver method (terminal fields). Nauta and Fink-Heimer methods (normal and degenerating fibers) and a method for AChE activity (cholinergic afferents). The transplants survived well in all combinations of donor and recipient ages used, and they survived and differentiated in all parts of the recipient brains, although relations to pial and ventricular surfaces appeared to be optimal. Cell differentiation continued after transplantation, and a characteristic laminar organization was retained, although least in embryonic donor tissues. The distribution of intrinsic connections was determined by the types of subfields present in the transplants and interaction with ingrown host afferents. All aberrant intrinsic connections observed corresponded to aberrant connections formed in the hippocampus and fascia dentata denervated in situ and included supragranular mossy fibers in the fascia dentata, aberrant infrapyramidal mossy fibers in CA3, spread of CA4-associated afferents beyond the normal commissural-associational zone in the dentate molecular layer together with ingrowth of CA3-associated and CA1-subiculum-associated afferents. Most transplants received a cholinergic input of host origin irrespective of the localization in the host brain, but also non-cholinergic host pathways innervated the transplants, in particular when the transplants were in close contact with host fiber tracts, and when the recipients were immature. At various transplant locations the non-cholinergic host afferents belonged to the commissural hippocampo-dentate system, the commissural hippocampal system and the callosal system. Other cases suggested innervation of dentate transplant by host entorhinal afferents. The formation and distribution of intrinsic transplant connections and connections between transplant and host appeared to be regulated by the same factors that regulate the development and reorganization of fiber connections in the normal and the in situ denervated hippocampus and fascia dentata. As a special variety of this, the distribution of cholinergic afferents adjusted to the distribution of the major intrinsic and extrinsic non-cholinergic pathways.

Distal infrapyramidal and longitudinal mossy fibers at a midtemporal hippocampal level

Timm's and horseradish peroxidase histochemical methods were used to demonstrate a small, distal infrapyramidal mossy fiber projection at midtemporal hippocampal levels in the rat. The Timm's sulfide silver method revealed sparse granules of dark, mossy fiber-like staining at an infrapyramidal location in an area roughly equivalent to hippocampal subfield CA3a (close to the CA1 border). The use of anterograde transport of horseradish peroxidase enabled the visualization of short mossy fiber axons that left the suprapyramidal bundle, penetrated the pyramidal cell layer, and terminated in a distal infrapyramidal position. The HRP-labeled axons possessed periodic swellings that are characteristic of mossy fiber morphology. The HRP studies also provided evidence for longitudinally oriented mossy fibers at midtemporal hippocampal levels. The distal tip of the suprapyramidal band of mossy fibers turned temporally and could be detected up to 1140 micrometers ventral to the main suprapyramidal band.

Lesion-induced sprouting of hippocampal mossy fiber collaterals to the fascia dentata in developing and adult rats.

A lesion-induced formation of an abnormal projection of hippocampal mossy fiber collaterals to the molecular layer of the fascia dentata was studied in rats. Both immature (1--30 days old) and adult rats were subjected to hippocampal and entorhinal lesions which alone or in combination removed one or more of the major afferents to the dentate molecular layer (commissural, associational, and perforant path). Some lesions in addition transected the main part of the mossy fibers en route from the dentate granule cells to the hippocampal pyramidal cells in regio inferior (CA3). The formation of aberrant mossy fiber terminals in fascia dentata (supragranular mossy fibers) was monitored by the histochemical Timm sulphide silver method, but the presence of aberrant terminals was also observed in the electron microscope. Abnormal amounts of supragranular mossy fiber terminals were found following entorhinal lesions of both immature and adult rats, but not following commissural lesions. Even larger amounts of aberrant terminals were, however, found in immature and adult rats subjected to lesions which removed most of the associational hippocampodentate projection by isolating columns of fascia dentata from major parts of the hilus (CA4). Pure transections of the fascia dentata perpendicular to its longitudinal septotemporal axis did not in itself cause aberrant supragranular terminals, although such lesions partially damaged the associational afferents. When the transections were combined with commissural lesions or entorhinal lesions or both, large amounts of supragranular terminals did, however, form at the denervated levels septal to the transection. After comparison of the amounts and distributions of the aberrant terminals found after the different lesions and in transplants of dentate tissue with different amounts of afferent input, we conclude that it is deafferentation of the dentate molecular layer, and not axotomy of the mossy fibers in the hilus of CA3 (pruning), that causes the aberrant growth of mossy fiber collaterals. Moreover, simultaneous removal of more than one afferent system seems to have a potentiating rather than a simple additive effect on the formation of supragranular mossy fibers.

Histochemical detection of zinc and copper in various neurons of the central nervous system

Trace elements (Zn2+, Cu2+, Fe2+) were localized with Timm's sulphide silver method in the neurons (pyramidal cells, Purkinje's cells, motoneurons) and in the axon terminals of various regions cortex, hippocampus, nucleus ruber, cerebellum, medulla spinalis) of the central nervous system. After treatment of the same tissues with 15% trichloroacetic acid, a considerable proportion of the coarse granular staining disappeared from certain neurons (Purkinje's cells, ncl. ruber cells, motoneurons) and axon terminals (hippocampus mossy fibres), while in other cells (cortex pyramidal cells) and axon terminals it remained. The results suggest that the decrease in the staining is a consequence of the loss of Zn2+ and Fe2+ from the neuronal perikaryon or axon terminal. The histochemical results are supported by quantitative atomic absorption measurements.

Genetic variation in the histoarchitecture of the hippocampal region of mice.

Timm's sulphide-silver method and the histochemical procedures for the demonstration of AChE, MAO and SDH were applied to the brains of three strains of adult mice of either sex: C57/BL/6J, DBA/2J and NMRI. Differences between strains were found 1) in the sulphide-silver pattern of the molecular layer of area dentata, probably reflecting differences in entorhinal, ipsilateral and/or commissural connections, 2) in the distribution of the mossy fibers, 3) in the AChE-staining of a suprapyramidal zone of regio inferior, probably reflecting differences in septal connections, 4) in the AChE-staining of the induseum griseum. The staining patterns for MAO and SDH did not vary, at least not qualitatively, between the strains investigated. Variation in adult age and sex did not influence the results. Since the differences observed seemed to reflect a pattern of genetic differentiation, five more inbred mice (A/J, AKR/A, BALB/c/A, C3H/Tif, St/6Fi) were included to strengthen the hypothesis that different genetic systems are operating at separate septo-temporal levels in the same areas during the development of the hippocampal formation.

Lesion-induced rerouting of hippocompal mossy fibers in developing but not in adult rats.

AbstractThe organization of the hippocampal mossy fiber system which projects from the granule cells of fascia dentata to the pyramidal cells of the hippocampal regio inferior (CA3) was studied after chronic lesions had been applied to CA3 in neonatal (0–3 day old), 5 and 21 day old, and adult rats. The distribution of terminals was monitored by the histochemical Timm sulphide silver method, and the fibers were demonstrated by the original Nauta silver method.Complete transections of CA3 in neonatal rats induced a layer of aberrant, infrapyramidal mossy fiber terminals in CA3 up to 1,300 μm septal to the transection followed by a corresponding expansion of the suprapyramidal mossy fiber layer for at least 720 μm. At increasing distance above the lesion, the induced changes moved laterally in CA3, affecting more distal parts of the mossy fibers. Corresponding to the layer of aberrant infrapyramidal terminals were aberrant bundles of mossy fibers, with an abnormally ascending septal course. No aberrant mossy fibers or terminals were induced temporal to the complete transections.Partial CA3 lesions in neonates induced changes corresponding to those observed after complete transections septal to the lesions. In addition, a layer of aberrant infrapyramidal mossy fiber terminals was induced in CA3 as far as 900 μm temporal to the partial lesions, accompained by an expansion of the suprapyramidal layer. The infrapyramidal terminals were related to aberrant bundles of fibers which followed an abnormally steep, temporal course, from which they in turn leveled off to join the suprapyramidal bundles.Compared to neonatal lesions, hippocampal transections performed on day 5 induced a slightly less dense layer of aberrant infrapyramidal mossy fiber terminals in CA3, and although a transection on day 21 still induced aberrant infrapyramidal terminals, these were far less abundant than after lesions at day 5. Transections in adult rats induced no such changes at all, and additional denervation of CA3 by simultaneous removal of the commisural projection from the contralateral hippocampus did not have any effect.The lesion‐induced, but age‐dependent redistribution of mossy fibers to CA3 is discussed in terms of reactive reinnervation following partial deafferentation, and compensatory sprouting and rerouting due to pruning‐like effects of the lesions. A rerouting of developing, not fully matured mossy fibers is found to be most likely: Deprived of normal target areas, the fibers have been forced to grow in abnormal directions, ending up hyperinnervating adjacent, accessible levels through the formation of aberrant infrapyramidal terminals and expansion of the suprapyramidal zone.

Localization of mercury in the rat ovary after oral administration of mercuric chloride

The Timm sulphide-silver method was used for histochemical localization of mercury in the ovaries of young rats fed with mercuric chloride. After 12 weeks of mercuric chloride administration test animals showed disturbances in the estrous cycle manifested by the prolongation of diestrous phase. In the ovaries of these rats the deposits of mercury were shown to occur particularly in macrophages of atretic young and mature ovarian follicles and corpora lutea, within granulosa cells of atretic follicles, and in the lutein cells of freshly formed corpora lutea. Follicular fluid and, irregularly, germinal epithelium also showed the ability to mercury accumulation. The ovaries of control animals treated according to the Timm procedure demonstrated negative histochemical reactivity.

Laminar differentiation of the hippocampus, fascia dentata and subiculum in developing rats, observed with the Timm sulphide silver method.

The laminar staining of the rat hippocampal region with the Timm sulphide silver method is from studies on adult rats known to depend on the various fibersystems terminating in these laminae. In order to illustrate the development of these fibersystems the laminar differentiation of the Timm staining of fascia dentata, hippocampus and subiculum is presented for rats between 1 and 31 days old. Corresponding changes in cytoarchitectonics as revealed by thionin staining are briefly demonstrated. Even on the first postnatal day there are indications of the mature, laminar staining pattern, and between three and nine days all the laminae corresponding to the terminal fields of the major afferent and intrinsic systems appear. After 12 days only minor additions to the laminar pattern develop, but there are adjustments of absolute and relative dimensions of layers and fields, and also the staining densities of individual laminae change. These observations are in good correlation with the available information on both hippocampal neurogenesis and cytodifferentiation, and the few fiber tracing studies performed on the developing hippocampal region. Compared to the latter, which ideally marks only one system or one lamina per animal, the Timm method provides an excellent means for getting an overview of the general developmental situation at the different ages. Thus developmental gradients along septotemporal, medio-lateral and basal-apical axes are found; which should be heeded in future studies on hippocampal synaptogenesis. The observations are discussed in relation to current models for neuronal growth and formation of nervous connections.

Concentration and Distribution of Heavy Metals in Nasal Mucosa of Nickel-Exposed Workers and of Controls, Studied with Atomic Absorption Spectrophotometric Analysis and with Timm's Sulphide Silver Method

Biopsy specimens from nasal mucosa of 30 nickel-exposed individuals and 6 controls were analysed by atomic absorption spectrophotometry to determine the content of nickel, copper, cobalt, zinc and iron. Timm's sulphide silver staining method was used for visualizing heavy metals in cryostat sections of biopsy material from each individual. The purpose of the investigation was to study the sulphide silver staining pattern in nasal mucosa of nickel-exposed individuals and controls, and to establish whether variations in heavy metal concentrations, especially nickel, affect the histochemical pattern in the mucosa. No consistent differences were found in the histochemical pattern between biopsies with high and low concentrations of nickel or any of the other metals. Some difference in epithelial types between specimens from the nickel-exposed group and the control group was seen, but the staining pattern was quite similar for corresponding epithelial types in the two groups. Two nasal carcinomas from nickel workers were virtually unstained with the sulphide silver staining method.

Concentration and Distribution of Heavy Metals in Nasal Mucosa of Nickel-Exposed Workers and of Controls, Studied with Atomic Absorption Spectrophotometric Analysis and with Timm's Sulphide Silver Method

Biopsy specimens from nasal mucosa of 30 nickel-exposed individuals and 6 controls were analysed by atomic absorption spectrophotometry to determine the content of nickel, copper, cobalt, zinc and iron. Timm's sulphide silver staining method was used for visualizing heavy metals in cryostat sections of biopsy material from each individual. The purpose of the investigation was to study the sulphide silver staining pattern in nasal mucosa of nickel-exposed individuals and controls, and to establish whether variations in heavy metal concentrations, especially nickel, affect the histochemical pattern in the mucosa. No consistent differences were found in the histochemical pattern between biopsies with high and low concentrations of nickel or any of the other metals. Some difference in epithelial types between specimens from the nickel-exposed group and the control group was seen, but the staining pattern was quite similar for corresponding epithelial types in the two groups. Two nasal carcinomas from nickel workers were virtually unstained with the sulphide silver staining method.

An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.

Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described. To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixature is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.

Visualization of the retino-hypothalamic projection in the rat by cobalt precipitation.

The technique of cobalt sulfide precipitation combined with Timm's sulfide-silver method for intensification of heavy metals was used to delineate the retino-hypothalamic projection of the rat. Freshly isolated rat brains were dissected and a solution of cobaltous chloride was applied to one of the cut optic nerves. Sixteen hours later, after cobalt ions had passed into the brain along the entire length of the optic fibers, the preparation was treated with ammonium sulfide to precipitate the cobalt as cobalt sulfide. In thick light microscopic sections, cobalt-filled axons were visualized as black fibers against a light gold background. Such fibers were observed to leave the posterior medial portion of the optic chiasm and, after arching dorsally, to project into the posterior fifth of the suprachiasmatic nucleus (SCN), as well as into the rostral part of the arcuate nucleus. Neither bifurcation of these axons nor looping of the axons back to the chiasm was seen. Most fibers projected to the SCN contralateral to the filled nerve, but the projection represented less than 0.1% of the total number of fibers in one optic nerve. These observations are considered to be graphic evidence of a retino-hypothalamic projection. The interpretation of the cobalt method is discussed, as are the functions of the connections that have been observed.

Cobalt sulphide staining of optic fibres in the brain of the cricket, Gryllus campestris.

Neuronal projections from one optic lobe to other parts of the brain were stained in the cricket Gryllus campestris using the cobalt sulphide technique and Timm's sulphide-silver method. The results are: Four tracts directly connect the medulla with the lobula and medulla of the contralateral optic lobe. Direct medullar projections end mainly in the non-glomerular neuropile of the protocerebrum, but also penetrate the calyx of the mushroom bodies, pons and central body in small numbers. A few somata which send fibres into the medulla lie in the pars intercerebralis, in the protocerebrum ventral to the opposite beta-lobe, the outer margin of the medulla of the contralateral optic lobe and between deuto- and tritocerebrum. The anatomical and physiological relevance of the stained connections is discussed.

Crossed pathways from the entorhinal area to the fascia dentata. II. Provokable in rats.

In the rat thhe perforant pathways from the entorhinal area normally innervate the fascia dentata only ipsilaterally. However, unilateral ablation of the entorhinal area (deentorhination) induces the formation of an anomalous crossed projection from the intact contralateral entorhinal area to the septal portion of the deafferented fascia dentata. After deentorhination of rats aged 1-30 days the organization of this projection was analyzed (a) by producing secondary lesions in the intact entorhinal area of perforant paths and observing the results anterograde degeneration with Fink-Heimer silver impregnation techniques, and (b) by staining with Timm's sulfide silver method whichmakes the terminal fields of afferent systems stand out in different tones of colors. Both methods showed the crossed entorhino-dentate projection to consist of two separable components. They were named the crossed medial perforant path and the crossed lateral perforant path, corresponding to their similarity in origin, dendritic localization of termination and Timm stainability to the ordinary, uncrossed medial and the lateral perforant pathways (MPP and LPP) which arise in the medial and lateral parts of the entorhinal cortex, respectively. Similarly induced crossed projections were demonstrated to the subcallosal continuation of fascia dentata, the fasciola cinerea. The heaviest terminal field of the crossed entorhino-dentate projection which was found in the most rostral and medial parts of the deafferented fascia dentata correlated with a lack of expected aberrant extension into theMPP and LPP terminal zones of commissural and ipsilateral hippocampodentate fibers. In Fink-Heimer preparations there was little variation in the distribution of the aberrant crossed sustems over the range of ages studied although the chronic operations performed earliest postnatally (5 days) tended to produce the heaviest representation. This latter observation appeared consistent with changes in the Timm staining pattern of the deafferented fascia dentata, since with an increase in age at the primary lesion from 5 to 14 days there was no increase in the spread into the fascia dentata of Timm stainable axon ter minals from CA3, interpreted as a sign of fewer crossed entorhinal afferents succeeding in a presumable competition with the CA3-derived system for available terminal space.

Histochemical observations on the permeation of heavy metals into taste buds of goldfish.

The present study was aimed at confirming whether metal-permeation into taste cells was responsible for the histological damage of taste buds of goldfish exposed to heavy metals. Histochemical observations employing TIMM's sulphide-silver method were made on the taste buds located in the palatal organs of goldfish exposed to 10-4M solutions of mercuric chloride, copper sulphate and zinc chloride, and 10-3M solutions of copper sulphate, zinc chloride and lead nitrate, Results revealed that the rate of metal-permeation into taste cells varied with the kind of metal, the concentration of the metallic ions in the medium, and the duration of exposure. In terms of permeation rate the sequence of metals was found to be mercury, copper, zinc and lead. Mercury damaged not only the taste buds but the entire epithelial layers within 30min. Lead affected mucous cells more than the taste buds. The results suggest that histological damage is associated with a ?? preceded by metal-permeation. During the early phase of exposure, mercury, copp ?? (10-4M) and zinc (10-3M) were found to permeate into one type of taste bud cell at ?? faster rate than into others.

Distribution of heavy metals in the hippocampal region of the guinea pig. A light microscope study with Timm's sulfide silver method.

With the present modification of Timm's sulfide silver method all parts of the hippocampal region show a distinctly stratified staining pattern, suggesting large regional differences in the content of heavy metals. The stain is largely confined to distinct grains, partly associated with neuronal somata and partly dispersed in the neuropil. Work in progress supports the idea that the grains in the neuropil are synaptic boutons, as has been shown previously for the mossy fibre layer. The staining pattern has been compared in detail with the fields and layers of the hippocampal region as delineated by cyto- and fibroarchitectonics. Previous concepts of the subdivision of this cortical region are confirmed and supplemented. The sulfide silver pattern of the guinea pig hippocampal region is fundamentally similar to that of the rat. However, the entorhinal area, the regio inferior hippocampi, and the dentate area show notable differences in the staining pattern between the two species.