Single Thin Sections Research Articles

3D topography of noncompact zone Golgi tubules in rat spermatids: A computer‐assisted serial section reconstruction study

Background In the Golgi apparatus, the 3D topography of saccules in the compact zones (CZs) is better understood than that of tubules in the noncompact zones (NCZs). The positioning of NCZ tubules relative to each other and to CZ saccules was studied in rat spermatids by computer-assisted serial section microscopy. Methods Twenty-four (semi) serials (3-6 consecutive sections each) in total were collected from untreated tissues and from tissues treated for glucose-6-phosphatase (G6P) cytochemistry as an alignment tool. The serials proceeded along either the cis-trans or the medial-lateral axes of the Golgi and collectively sampled all portions of this organelle. Selected serials were computer reconstructed and the final models displayed in red-green/red-blue stereo. Results In single thin sections, NCZ tubules typically appeared randomly oriented; however, in serial sections a high degree of organization was evident. Most tubules were traceable to the type of tubular networks (TNs) that interconnect equivalent CZ saccules (see review Rambourg and Clermont, 1990) Such TNs were present at consecutive saccular levels through each NCZ, were stacked like the saccules from which they originate, and in many regions were aligned from cis-trans. The cis-most of the TNs projected above the cis-pole of the stacked saccules and were penetrated by coated and uncoated ER buds. Conclusions The function of the extensive NCZ tubular domain, consisting of the stacked and aligned TNs, will have to be addressed in future studies. However, the specific topography of the cis-most TNs make them candidates to serve as acceptor membranes in ER-Golgi transport. Anat. Rec. 250:381–396, 1998. © 1998 Wiley-Liss, Inc.

3D topography of noncompact zone Golgi tubules in rat spermatids: a computer-assisted serial section reconstruction study.

In the Golgi apparatus, the 3D topography of saccules in the compact zones (CZs) is better understood than that of tubules in the noncompact zones (NCZs). The positioning of NCZ tubules relative to each other and to CZ saccules was studied in rat spermatids by computer-assisted serial section microscopy. Twenty-four (semi) serials (3-6 consecutive sections each) in total were collected from untreated tissues and from tissues treated for glucose-6-phosphatase (G6P) cytochemistry as an alignment tool. The serials proceeded along either the cis-trans or the medial-lateral axes of the Golgi and collectively sampled all portions of this organelle. Selected serials were computer reconstructed and the final models displayed in red-green/red-blue stereo. In single thin sections, NCZ tubules typically appeared randomly oriented; however, in serial sections a high degree of organization was evident. Most tubules were traceable to the type of tubular networks (TNs) that interconnect equivalent CZ saccules (see review Rambourg and Clermont, 1990) Such TNs were present at consecutive saccular levels through each NCZ, were stacked like the saccules from which they originate, and in many regions were aligned from cis-trans. The cis-most of the TNs projected above the cis-pole of the stacked saccules and were penetrated by coated and uncoated ER buds. The function of the extensive NCZ tubular domain, consisting of the stacked and aligned TNs, will have to be addressed in future studies. However, the specific topography of the cis-most TNs make them candidates to serve as acceptor membranes in ER-Golgi transport.

Dual character, asynaptic and synaptic, of the dopamine innervation in adult rat neostriatum: A quantitative autoradiographic and immunocytochemical analysis

Dopamine (DA) axon terminals (varicosities) in the neostriatum of adult rats were examined for shape, size, content, synaptic incidence, type of junction, synaptic targets, and microenvironment after electron microscopic identification either by [3H]DA uptake autoradiography or by immunocytochemistry with monoclonal antibodies against DA-glutaraldehyde-protein conjugate. Both approaches yielded comparable results. Whether they were from the paraventricular or the mediodorsal neostriatum, respectively, the [3H]DA-labeled and DA-immunostained varicosities were generally oblong and relatively small; more than 60% contained one or more mitochondria. Sixty to seventy percent were asynaptic, and 30–40% were endowed with a synaptic membrane differentiation (junctional complex), as inferred by stereological extrapolation from single thin sections (both approaches) or observed directly in long, uninterrupted series of thin sections (immunocytochemistry). The synaptic DA varicosities always displayed symmetrical junctions: 67% with dendritic branches, 30% with dendritic spines, and 2–3% with neuronal cell bodies. DA varicosities juxtaposed to one another were frequent. Other axonal varicosities were more numerous in the immediate vicinity of DA varicosities than around randomly selected, unlabeled terminals. The respective microenvironments of DA and unlabeled varicosities also showed enrichment in the preferred synaptic targets of both groups of varicosities, with dendritic branches for DA and dendritic spines for the unlabeled ones. These data suggest a dual mode of operation that is diffuse as well as synaptic for the nigrostriatal DA system. In such a densely DA-innervated brain region, they also dead to the hypothesis that a basal level of extracellular DA might be maintained permanently around every tissue constituent and, thus, contribute to the mechanisms of action, properties, and functions (or dysfunctions) of DA within the neostriatum itself and as part of the basal ganglia circuitry. © 1996 Wiley-Liss, Inc.

Dual character, asynaptic and synaptic, of the dopamine innervation in adult rat neostriatum: a quantitative autoradiographic and immunocytochemical analysis.

Dopamine (DA) axon terminals (varicosities) in the neostriatum of adult rats were examined for shape, size, content, synaptic incidence, type of junction, synaptic targets, and microenvironment after electron microscopic identification either by [3H]DA uptake autoradiography or by immunocytochemistry with monoclonal antibodies against DA-glutaraldehyde-protein conjugate. Both approaches yielded comparable results. Whether they were from the paraventricular or the mediodorsal neostriatum, respectively, the [3H]DA-labeled and DA-immunostained varicosities were generally oblong and relatively small; more than 60% contained one or more mitochondria. Sixty to seventy percent were asynaptic, and 30-40% were endowed with a synaptic membrane differentiation (junctional complex), as inferred by stereological extrapolation from single thin sections (both approaches) or observed directly in long, uninterrupted series of thin sections (immunocytochemistry). The synaptic DA varicosities always displayed symmetrical junctions: 67% with dendritic branches, 30% with dendritic spines, and 2-3% with neuronal cell bodies. DA varicosities juxtaposed to one another were frequent. Other axonal varicosities were more numerous in the immediate vicinity of DA varicosities than around randomly selected, unlabeled terminals. The respective microenvironments of DA and unlabeled varicosities also showed enrichment in the preferred synaptic targets of both groups of varicosities, with dendritic branches for DA and dendritic spines for the unlabeled ones. These data suggest a dual mode of operation that is diffuse as well as synaptic for the nigrostriatal DA system. In such a densely DA-innervated brain region, they also lead to the hypothesis that a basal level of extracellular DA might be maintained permanently around every tissue constituent and, thus, contribute to the mechanisms of action, properties, and functions (or dysfunctions) of DA within the neostriatum itself and as part of the basal ganglia circuitry.

Ultrastructural relationships between noradrenergic nerve fibers and non-neuronal elements in the rat cerebral cortex

Pharmacological and biochemical data suggest that noradrenaline (NA)-containing fibers not only regulate the activity of cortical neurons but also influence the functional state of non-neuronal elements. In the present study, immunocytochemistry with an antiserum against NA, followed by silver-gold intensification of the immunoreaction end-product, was employed to examine the ultrastructural relationships between the NA fiber system and the intraparenchymal blood vessels, oligodendrocytes, and astrocytes in the rat visual cortex. Electron microscopy revealed a large number of fine varicose NA fibers to be in intimate contact with cortical capillaries. Examination of single thin sections showed that NA boutons were usually separated from the capillary wall by a fine astroglial sleeve. However, serial section analysis revealed that the continuity of the astrocytic end feet was interrupted at sites, resulting in direct apposition of the perivascular NA fibers to the capillary basal lamina. Noradrenergic fibers were found to contact both types of macroglial cells. Single or clustered oligodendrocytes in intimate contact with NA fibers were observed throughout the cortical depth. Individual contacts could be followed in more than six successive thin sections, and oligodendrocyte plasma membrane frequently exhibited a light thickening at the sites of the NA fiber apposition. NA fiber-astroglial relationships were largely encountered in supragranular layers. In these layers, astrocytic cell bodies were characteristically outlined by fine varicose NA fibers. However, no plasma membrane differentiations were observed at the sites of intimate NA fiber apposition. The present ultrastructural findings provide the anatomical substrate for the control exerted by the NA fiber system over cortical microvasculature and macroglia.

An ultrastructural study of cholinergic and non-cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei in the rat.

Synaptic connectivity and other ultrastructural features of cholinergic and non-cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were investigated with electron microscopy combined with pre-embedding immunohistochemistry for choline acetyltransferase. Quantitative morphometric analyses were conducted on selected immunopositive as well as immunonegative neurons. The ultrastructure of immunoreactive neurons in the laterodorsal and pedunculopontine tegmental nuclei was similar. In both nuclei, immunoreactive neurons were among the larger neurons, and somatic areas of immunopositive neurons in single thin sections were larger than those of immunonegative neurons by an average of 40%. Immunopositive somata varied in shape, appearing polygonal, fusiform or oval. Regardless of immunoreactivity, however, neurons in the pedunculopontine nucleus tended to have more irregular shapes than those in the laterodorsal tegmental nucleus. Immunoreactive neurons in both the nuclei had abundant cytoplasmic organelles and a large, clear nucleus with a few infoldings. Usually, about a quarter of the surface of an immunopositive soma was covered with astrocytic processes, and some immunopositive somata were directly apposed to an astrocyte. Immunoreactive dendrites and, less frequently, axon terminals were seen in close apposition to endothelial cells of blood capillaries or pericytes. Immunoreactive somata and dendrites in the laterodorsal and pedunculopontine tegmental nuclei received many synapses, mainly from unlabelled axon terminals. The mean number (4.7 +/- 1.8) of synapses received by immunolabelled somata in single thin sections was greater, by about 70%, than those received by unlabelled somata. The presynaptic axon terminals synapsing with immunoreactive somata commonly contained small, round and clear vesicles, and 20% of them contained a few dense-cored vesicles as well. Immunoreactive dendrites, in addition, received synapses from unlabelled axon terminals containing flat and clear vesicles, which accounted for 15% of the synapses with immunoreactive dendrites. Many immunopositive axon terminals were present in both the tegmental nuclei. They contained clear round vesicles, and usually synapsed with unlabelled dendrites. A few immunolabelled axons, however, appeared to synapse with immunopositive somata and dendrites. Immunoreactive fibres were also present in both the tegmental nuclei. They were either thinly myelinated or unmyelinated. In conclusion, the ultrastructural morphology of cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei is similar, and these neurons represent a distinct population of neurons in both nuclei in that they are larger and receive more synaptic contacts than non-cholinergic neurons. Cholinergic neurons, however, appear to receive synapses from cholinergic axon terminals only rarely, despite the abundance of cholinergic terminals in the tegmental nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Septohippocampal neurons in the rat septal complex have substantial glial coverage and receive direct contacts from noradrenaline terminals

The ultrastructure of septohippocampal neurons in the septal complex and their relations with catecholamine, principally noradrenaline, terminals were examined in single thin sections. Projection neurons were identified by retrograde transport of wheat-germ agglutinated apo-horseradish peroxidase conjugated to colloidal gold particles (WAHG) following an injection into the hippocampal formation of anesthetized adult rats. After a 1 day survival, sections through the septal complex were labeled with antibodies to tyrosine hydroxylase (TH) or dopamine-β-hydroxylase (DBH). By light microscopy, numerous processes with TH- and DBH-immunoreactivity were near neurons containing retrogradely transported WAHG. By electron microscopy, most WAHG was associated with lysosomes, multivesicular and ‘sequestration’ bodies in the cytoplasm of perikarya and large dendrites. WAHG-labeled perikarya ( n = 114) had a large amount of astrocytic coverage (> 60% of surface) and a low amount of terminal coverage (< 25%). WAHG-labeled perikarya and dendrites were either directly contacted by TH- or DBH-labeled terminals or abutted glial processes apposed to TH- or DBH-labeled terminals. Immunoreactivity for TH and DBH was found primarily in axons and axon terminals. The morphology and synaptic associations of TH-labeled terminals was similar to that reported previously. DBH-labeled terminals ( n = 314; 0.5 ± 0.2 μm in diameter) contained numerous small clear vesicles and from 0–4 large, dense-core vesicles. DBH-containing terminals: (1) contacted perikarya and dendrites (58%), 10% of which contained WAHG; (2) were closely apposed to other terminals (7%); or (3) were separated by glial processes (35%). DBH-labeled terminals formed chiefly symmetric synapses on perikarya. However, most DBH-containing terminals formed both asymmetric and symmetric synapses on the shafts of small dendrites, suggesting both excitatory and inhibitory functions for noradrenaline terminals on septal neurons. The results demonstrate that septohippocampal neurons (1) are mostly engulfed by astrocytes and have very little terminal coverage; (2) are both directly contacted (synapses) and indirectly contacted (appositions to opposing astrocytes or axon terminals) by catecholamine, particularly noradrenaline, terminals.

Reaction textures and fluid behaviour in very high pressure calc‐silicate rocks of the Münchberg gneiss complex, Bavaria, Germany

ABSTRACTCalc‐silicate rocks occur as elliptical bands and boudins intimately interlayered with eclogites and high‐pressure gneisses in the Münchberg gneiss complex of NE Bavaria. Core assemblages of the boudins consist of grossular‐rich garnet, diopside, quartz, zoisite, clinozoisite, calcite, rutile and titanite. The polygonal granoblastic texture commonly displays mineral relics and reaction textures such as post kinematic grossular‐rich garnet coronas. Reactions between these mineral phases have been modelled in the CaO‐Al2O3‐SiO2‐CO2‐H2O system with an internally consistent thermodynamic data base. High‐pressure metamorphism in the calc‐silicate rocks has been estimated at a minimum pressure of 31 kbar at a temperature of 630d̀ C with XH2, O ≥ 0.03. Small volumes of a CO2‐N2‐rich fluid whose composition was buffered on a local scale were present at peak‐metamorphic conditions. The P‐T conditions for the onset of the amphibolite facies overprint are about 10 kbar at the same temperature. XCo2 of the H2O‐rich fluid phase is regarded to have been &lt;0.03 during amphibolite facies conditions. These P‐T estimates are interpreted as representing different stages of recrystallization during isothermal decompression. The presence of multiple generations of mineral phases and the preservation of very high‐pressure relics in single thin sections preclude pervasive post‐peak metamorphic fluid flow as a cause of a re‐equilibration within the calc‐silicates. The preservation of eclogite facies, very high‐pressure relics as well as amphibolite facies reactions textures in the presence of a fluid phase is in agreement with fast, tectonically driven unroofing of these rocks.

Ultrastructural and morphometric features of the acetylcholine innervation in adult rat parietal cortex: an electron microscopic study in serial sections.

This study was aimed at characterizing the ultrastructural morphology of the normal acetylcholine (ACh) innervation in adult rat parietal cortex. After immunostaining with a monoclonal antibody against purified rat brain choline acetyltransferase (ChAT), more than 100 immunoreactive axonal varicosities (terminals) from each layer of the Par 1 area were photographed and examined in serial thin sections across their entire volume. These varicosities were relatively small, averaging 0.6 micron in diameter, 1.6 microns 2 in surface, and 0.12 micron 3 in volume. In every layer, a relatively low proportion exhibited a synaptic membrane differentiation (10% in layer I, 14% in II-III, 11% in IV, 21% in V, 14% in VI), for a I-VI average of 14%. These synaptic junctions were usually single, symmetrical (> 99%), and occupied a small portion of the surface of varicosities (< 3%). A majority were found on dendritic branches (76%), some on spines (24%), and none on cell bodies. On the whole, the ACh junctional varicosities were significantly larger than their nonjunctional counterparts, and both synaptic and nonsynaptic varicosities could be observed on the same fiber. A subsample of randomized single thin sections from these whole varicosities yielded similar values for size and synaptic frequency as the result of a stereological extrapolation. Also analyzed in single sections, the microenvironment of the ChAT-immunostained varicosities appeared markedly different from that of unlabeled varicosity profiles randomly selected from their vicinity, mainly due to a lower incidence of synaptically targeted dendritic spines. Thus, the normal ACh innervation of adult rat parietal cortex is predominantly nonjunctional (> 85% of its varicosities), and the composition of the microenvironment of its varicosities suggests some randomness in their distribution at the microscopic level. It is unlikely that these ultrastructural characteristics are exclusive to the parietal region. Among other functional implications, they suggest that this system depends predominantly on volume transmission to exert its modulatory effects on cortical activity.

Rapid development of somatic spines in stratum granulosum of the adult hippocampus in vitro

Somatic spines on granule cells have been observed occasionally in the adult rat dentate gyrus in vivo. Here we evaluate the appearance and formation of somatic spines in s. granulosum from adult rat hippocampal slices immediately after slice preparation, 45 min, 3 h, and 5 h later. Initially somatic spines are extremely rare but, after 3 h in vitro, they are readily apparent. Some of these somatic spines form asymmetric synapses that have a spherical vesicle-containing presynaptic bouton and a postsynaptic density. Other somatic spines lack a postsynaptic density and may also lack an apposed presynaptic bouton (free somatic spines) as observed in single thin sections. Both the number of these free somatic spines and the number of somatic spine synapses increase in a time-dependent manner. No somatic spines were observed on CA1 pyramidal cells in the same hippocampal slices. Electrophysiological observations indicate that the formation of somatic spine synapses on granule cells in the hippocampal slice occurs without any apparent granule cell activation. The trigger event for this very rapid synaptogenesis in the adult dentate gyrus remains to be determined.

Petrological evolution of amphibolite shear zones, Cheyenne Belt, south‐eastern Wyoming, USA

Abstract Metre‐scale amphibolite boudins in the Cheyenne Belt of south‐eastern Wyoming are cut and deformed by shear zones which preserve a full strain transition across 7 cm, from relatively undeformed amphibolite with a relict igneous texture to mylonitic amphibolite with an L‐S tectonic fabric. The strain transition is marked by the progressive rotation of amphibole + plagioclase aggregates into parallelism with the shear‐zone boundary. An increase in strain magnitude is indicated by development of the tectonic fabric and progressive reduction of amphibole and plagioclase grain size as a result of cataclasis. Bulk chemistry of five samples across a single strain transition shows no significant or systematic variation in major element chemistry except for a minor loss of SiO2, which indicates that the shear zone was a system essentially closed to non‐volatile components during metamorphism and deformation. Amphibolites throughout the shear zone consist of amphibole and plagioclase with only minor amounts of quartz, chlorite, epidote, titanite and ilmenite. Within the relatively undeformed amphibolite, amphibole and plagioclase have wide compositional ranges in single thin sections. Amphibole compositions vary from actinolitic hornblende to magnesio‐hornblende with increases in Al, Fe, Na and K contents and decreases in Si and Mg that can be modelled as progress along tschermakite, edenite and FeMg‐1 exchange vectors from tremolite. Plagioclase ranges from An60 in cores to An30 within grain‐boundary domains. With increasing strain magnitude, local variation of amphibole composition decreases as amphibole becomes predominantly magnesio‐hornblende. Plagioclase composition range also decreases, although grain‐boundary domains still have higher albite content. These petrological data indicate that shear‐zone metamorphism was controlled by the magnitude of strain during synmetamorphic deformation. SEM and microprobe imaging indicate that chemical reactions occurred by a dissolution and reprecipitation process during or after cataclastic deformation. This suggests that grain‐boundary formation was an important process in the petrological evolution of the shear zone, possibly by providing zones for fluid ingress to facilitate metamorphic reactions. These results highlight the necessity for conducting detailed microstructural evaluation of rocks in order to interpret petrological, isotopic and geochronological data.

Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: implications for the maturation of synaptic physiology and long-term potentiation [published erratum appears in J Neurosci 1992 Aug;12(8):following table of contents

It has long been hypothesized that changes in dendritic spine structure may modify the physiological properties of synapses located on them. Due to their small size, large number, and highly variable shapes, standard light microscopy of Golgi impregnations and electron microscopy (EM) of single thin sections have not proved adequate to identify most spines in a sample or to quantify their structural dimensions and composition. Here we describe a new approach, the series sample, that was developed to classify by shape and subcellular composition all of the spines and synapses in a sample of neuropil by viewing them through serial EM sections. Spines in each class are then randomly selected for serial reconstruction and measurement in three dimensions. This approach was used to assess whether structural changes in hippocampal CA1 spines could contribute to the enhanced synaptic transmission and the greater endurance of long-term potentiation (LTP) that occur with maturation. Our results show a near doubling in the total density of synapses in the neuropil and along reconstructed dendrites between postnatal day 15 (PND 15) and adult ages. However, this doubling does not occur uniformly across all spine and synapse morphologies. Thin spines, mushroom spines containing perforated postsynaptic densities (PSDs) and spine apparatuses, and branched spines increase by about four-fold in density between PND 15 and adult ages. In contrast, stubby spines decrease by more than half and no change occurs in mushroom spines with macular PSDs or in dendritic shaft synapses. The stubby spines that remain are smaller in adults than at PND 15 and the mushroom spines are larger, while no change occurs in the three-dimensional structure of thin spines. Only a few spine necks at either age are constricted or long enough to attenuate charge transfer; therefore, the doubling in synapses should mediate the enhancement of synaptic transmission that occurs with maturation. In addition, LTP is not likely to be mediated by widening of spine necks at either age. However, the constricted spine necks could serve to concentrate specific molecules at activated synapses, thereby enhancing the specificity and endurance of LTP with maturation. These results demonstrate that the new series sample method combined with three-dimensional reconstruction reveals quantitative changes in the frequency and structure of spines and synapses that are not discernable by other methods and are likely to have dramatic effects on synaptic physiology and plasticity.

Synaptic organization of the serotoninergic input to the superficial gray layer of the hamster's superior colliculus.

Immunocytochemistry with an antibody directed against a serotonin (5-HT)-bovine serum albumin (BSA) conjugate was combined with electron microscopy and serial section analysis to examine the synaptic organization of the serotoninergic projection to the stratum griseum superficiale (SGS) of the hamster's superior colliculus (SC). Most of the 5-HT-immunoreactive swellings in the hamster's SC did not make conventional synaptic contacts. Examination of 250 such swellings in single thin sections revealed only 11 synapses. Assessment of an additional 50 swellings followed through serial sections showed that only 6% (N = 3) of these varicosities made conventional synaptic contacts. All the synapses made by serotoninergic axons were with the dendrites of SC neurons. These results demonstrate that the inhibitory effects of 5-HT on SC neurons in hamster are mainly mediated by the nonsynaptic release of this indoleamine.

Ultrastructural analysis of the serotonin hyperinnervation in adult rat neostriatum following neonatal dopamine denervation with 6-hydroxydopamine

Serotonin (5-HT) immunocytochemistry was used at the electron microscopic level to characterize the ultrastructural features of 5-HT axon terminals (varicosities) hyperinnervating the neostriatum of adult rats, 3 months after a neonatal destruction of the nigrostriatal dopamine system by intraventricular 6-hydroxydopamine. 5-HT-immunostained terminals from the anterior half of the hyperinnervated neostriatum were examined in single thin sections, and compared to their counterparts in vehicle-injected controls with respect to shape, size, organelle content, presence of a synaptic membrane differentiation and composition of the microenvironment. The intrinsic and relational features of the 5-HT-immunostained varicosities were essentially the same in 5-HT-hyperinnervated and control tissue. In particular, the frequency with which these varicosities made synaptic contacts was similarly low in both conditions (6–8% for whole varicosities), as already described in normal adult rat neostriatum. The distributional frequency of elements juxtaposed to the 5-HT-immunostained varicosities was also comparable in control and 5-HT-hyperinnervated tissue. However, in both conditions, there were much fewer dendritic spines in the microenvironment of 5-HT varicosities than around unlabeled terminals randomly selected from the same thin sections. This difference seemed entirely due to the numerous axo-spinous synaptic contacts made by the randomly selected, unlabeled varicosities. Together with recent observations on the 5-HT-hyperinnervation of adult rat hippocampus after grafts of fetal neurons, these data lead to the suggestion that mostly non-junctional neostriatal 5-HT terminals are not committed to a specific intratissular microenvironment. This might in part explain why they grow in excess when reinnervating adult tissue after a lesion or a graft.

Transcapillary transport of solute by the endothelial vesicular system: Evidence from thin serial section analysis

Upon crossing the walls of continuous capillaries, perfused electron-dense solutes often appear to become lodged in the attenuated space bounded by pericytes and the ablumenal capillary surface. In single thin sections, it is difficult to determine whether such accumulations result from transcellular transport via the endothelial vesicular system or whether tracer has filled these spaces retrogradely after paracellular transport through interendothelial clefts. We have taken continuous thin serial sections through patches of terbium trapped between pericytes and the endothelial cells of continuous capillaries in the rete mirabile of the eel. Sampling these areas in three dimensions has revealed that the terbium deposits are bounded and limited to regions of the capillary wall devoid of interendothelial associations. Dense interstitial terbium deposits were also continuous with ablumenal endothelial caveolae and could also be seen in clusters of fused vesicles. These observations strongly implicate transcellular transport by the endothelial vesicular system as the route by which accumulated terbium had crossed the capillary wall.

Formation of the tetraploid intermediate is associated with the development of cells with more than four centrioles in the elastase-simian virus 40 tumor antigen transgenic mouse model of pancreatic cancer.

The development of pancreatic cancer in transgenic mice expressing the simian virus 40 tumor antigen placed under controlling regions of the elastase I gene is characterized by the sequential appearance of tetraploid and then multiple aneuploid cell populations. Pancreatic tissues from such transgenic mice were studied between 8 and 32 days of age. Virtually 100% of acinar cell nuclei had immunohistochemically detectable tumor antigen by 18 days. Tetraploid cells were demonstrated by DNA content flow cytometry by 20 days and were associated with the appearance of interphase cells that had 5-11 centrioles per cell in single thin sections of pancreatic tissue examined by electron microscopy. Mitotic cells also were observed that had 5 or more centrioles per cell that were incorporated into the poles of bipolar or at least tripolar spindle apparatuses. These observations indicate that formation of the tetraploid intermediate in the diploid----tetraploid----aneuploid sequence of pancreatic tumor formation in elastase-simian virus 40 tumor antigen transgenic mice is accompanied by the development of cells with 5 or more centrioles that can be incorporated into the poles of abnormal mitotic spindles. We speculate that cells with more than 4 centrioles are predisposed to the formation of multipolar mitoses that may yield daughter cells with chromosomal gains and losses, resulting in the subsequent development of aneuploid tumors.

Ultrastructural features of the serotonin innervation in adult rat hippocampus: An immunocytochemical description in single and serial thin sections

This study was aimed at characterizing the fine-structural features of the normal serotonin (5-HT) innervation in adult rat hippocampus, by means of electron microscopic immunocytochemistry with a polyclonal antiserum against 5-HT-glutaraldehyde-protein conjugate (donated by Michel Geffard, Bordeaux). Two hippocampal sectors were examined, at mid-level along the septo-temporal axis: CA3-a of Ammon's horn and crest of the dentate gyrus (DG-c). A large number of axonal varicosities (terminals) were sampled in single ultrathin sections, to achieve a statistically significant comparison of their size and of their relative frequency of synaptic specialization, junctional targets and juxtaposed elements, between the oriens and the radiatum layer of CA3-a, and the molecular and the polymorph layer of DG-c. In both CA3-a layers, the microenvironment of the immunostained terminals was also compared to that of a population of unlabeled varicosities randomly selected from the same micrographs. Moreover, 57 varicosities from the oriens and the radiatum layer of CA3-a were visualized in a long series of thin sections, allowing for their examination from end to end in 43 instances. As measured in single sections, hippocampal 5-HT varicosities were of comparable diameter (0.57 μm on the average) in the two anatomical sectors and four neuropil layers examined. As extrapolated stereologically to whole varicosities, the proportion making a synaptic membrane specialization (synaptic incidence) ranged from 18 to 33% (average of 24%), without statistically significant differences between the two sectors and four layers. The synaptic incidence determined directly from serial sections of CA3-a (18%) was nearly identical to that extrapolated from single sections (18.1% in the oriens and 19.5% in the radiatum layer). In both CA3-a and DG-c, the 5-HT varicosities showing a junctional complex were slightly larger than their non-junctional counterparts. In CA3-a, only dendritic shafts were targeted by synaptic 5-HT varicosities, whereas in DG-c there were also a few axo-spinous synapses. The microenvironment of CA3-a 5-HT varicosities differed markedly from that of randomly selected unlabeled varicosities, due to its much lower frequency of synaptic targets and higher frequency of juxtaposed axonal varicosities, at least in the radiatum layer. In all four layers examined, other axonal varicosities were indeed the most frequently encountered neuronal element in the immediate vicinity of immunostained 5-HT varicosities. Neurites and dendritic shafts were also common, but dendritic spines (4%) were relatively infrequent. Thus, the normal 5-HT innervation of adult rat hippocampus is predominantly non-junctional (70–80% of its varicosities), and it exhibits comparable intrinsic and relational features in the four neuropil layers of CA3-a and DG-c.

GABA innervation in adult rat oculomotor nucleus: a radioautographic and immunocytochemical study.

GABA innervation in the adult rat oculomotor nucleus (n.III) was investigated using two complementary approaches: radioautography after incubation of brain slices with tritiated GABA ([3H]GABA) along with local in vivo microinjections of the tracer, and GABA immunocytochemical procedures involving antibodies directed against a GABA-glutaraldehyde-protein conjugate. As determined by radioautography after in vitro or in vivo labelling, the [3H]GABA uptake sites in the n.III mainly involved axon terminals. These were distributed throughout the neuropil and were often closely apposed to unlabelled motoneuron somata. A small number of glial cells also showed preferential accumulation of the tracer. The GABA-immunostaining likewise involved axon terminals throughout the nucleus, but no glial cells were immunopositive. In the dorsal region of the structure, occasional GABA-immunostained internuclear neurons were observed among unstained motoneuron cell bodies. Electron microscopic examination of [3H]GABA-labelled or GABA-immunostained profiles in n.III revealed axon terminals of around 1 micron in diameter, always filled with small, round synaptic vesicles homogeneously distributed throughout the axoplasm. These boutons frequently contained mitochondria and one or more large granular vesicles. In single thin sections, 35% of [3H]GABA-labelled, and 19% of GABA-immunostained varicose profiles exhibited a synaptic differentiation, suggesting the existence of a predominantly if not entirely junctional innervation. These synapses mostly involved dendritic trunks or dendritic branches and were usually of the symmetrical type. A few, which were always symmetrical, were also observed on large somata of motoneurons. Some of the dendrites synaptically contacted by GABA-immunostained axon terminals were themselves GABA immunoreactive. These data substantiate the idea that GABA is involved in the control of motoneuron activity in n.III, and provide a structural basis for the inhibitory role of this transmitter in oculomotor function.

Serotonin innervation in adult rat neostriatum. II. Ultrastructural features: a radioautographic and immunocytochemical study

High-resolution radioautography after cerebroventricular administration of tritiated serotonin (5-HT) and PAP immunocytochemistry with an antiserum against 5-HT-glutaraldehyde conjugate (kindly donated by M. Geffard) were used in parallel to investigate the intrinsic and relational fine structural features of 5-HT axon varicosities (terminals) in the neostriatum of the adult rat. The uptake-labeled varicosities were examined in single thin sections from a paraventricular sector of neostriatum, whereas their immunostained counterparts were viewed in serial thin sections form the same paraventricular sector plus a dosal neostrial sector. The two approaches yielded complementary results in terms of varicosity dimensions, synaptic features and appositional relationships. Serotonin axon terminals were generally small and, as measured in immunostained material, even smaller in the dorsal than in the paraventricular neostriatum. Their internal features, best viewed in radioautographs, included small pleomorphic synaptic vesicles with occasional large granular vesicles and mitochondria. Junctional 5-HT terminals from both the paraventricular and the dorsal neostriatal sectors synapsed exclusively, and with equal frequency, on dendritic spines or shafts, almost always with asymmetrical membrane differentiations. The proportion of junctional varicosities, however, was very low in serial (immunocytochemical) as well as single (radioautographic) thin sections. Only 10–13% of 5-HT varicosities from either the paraventricular or the dorsal neostriatum exhibited a synaptic junction, in contrast with a junctional incidence of at least 70% for randomly selected axonal varicosities similarly sampled in the surrounding neuropil. Serotonin axon terminals, whether or not synaptic, were closely apposed to a variety of structures comprising mostly other axon terminals, dendritic spines and branches, but rarely neuronal somata. The synaptic and appositional features of immunostained 5-HT varicosities were similar for both the dorsal and the paraventricular neostriatum. In this context, it is likely that the effects of 5-HT in the neostriatum are exerted upon a multiplicity of cellular target sites in addition to the restricted number of dendritic spines and shafts synaptically contacted by this type of monoamine terminal.

Serotonin axon terminals in the ventral tegmental area of the rat: fine structure and synaptic input to dopaminergic neurons

The serotoninergic (5-hydroxytryptamine, 5-HT) innervation of the rat ventral tegmental area (VTA) was examined by light and electron microscopic radioautography following intraventricular infusion of [ 3H]5-HT. The [ 3H]5-HT labeled processes were characterized with respect to their regional distribution, ultrastructure and relationships with all neurons, including dopaminergic neurons, identified in the same sections using immunocytochemistry for the localization of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). By light microscopy, [ 3H]5-HT labeled axons and axonal varicosities were detected throughout the interfascicular nucleus and ventral portion of the VTA. By electron microscopy, [ 3H]5-HT-labeled axons were found to be mainly small and unmyelinated, although a few showed several lamellae of myelin. The labeled varicosities measured 0.6 μm in mean diameter and contained many small, round or flattened agranular vesicles and a few large granular vesicles. More than 18% showed synaptic specializations in single thin sections. Most of these synapses were asymmetric and established on dendritic shafts. Based on the probability of seeing such synaptic specializations in single thin sections, it was estimated that as many as 50% of the labeled 5-HT terminals formed synaptic contacts in the VTA. In dually labeled light microscopic sections, [ 3H]5-HT-accumulating processes often appeared adjacent to TH-immunoreactive perikarya and proximal dendrites. Electron microscopy demonstrated that terminals with radioautographic labeling for 5-HT formed conventional synapses both with TH-labeled and unlabeled dendrites in the VTA. Many additional 5-HT terminals lacking recognizable synaptic densities were directly apposed to TH-labeled dendrites and were isolated from the rest of the neuropil by thin glial leaflets. These results suggest that 5-HT neurons innervate both dopaminergic and non-dopaminergic neurons in the VTA and may influence mesocortical and mesolimbic efferent systems through synaptic as well as non-synaptic mechanisms.