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The binding of Bcl-2 to Beclin-1 reduces Beclin-1's capacity to induce autophagy. Here, we have tested whether the interaction is reciprocated by loss of Bcl-2's anti-apoptotic function. We targeted Bcl-2 to mitochondria or endoplasmic reticulum (ER) and induced apoptosis using several apoptotic stimuli that initiate ER and/or mitochondrial signaling pathways (UV radiation, TNF and cycloheximide, staurosporine, thapsigargin and tunicamycin). When Beclin-1 and Bcl-2 were expressed together in HeLa cells, Beclin-1 (but not Beclin-1 lacking the Bcl-2-binding domain) followed Bcl-2 to the appropriate organelle with complete or near-complete overlap (comprising 60 and 30% of cells, respectively). The interaction between Beclin-1 and Bcl-2 was verified by immunoprecipitation, and a membrane-proximate localization of Beclin-1 was shown by immunoelectron microscopy. Apoptosis was followed by measuring changes in nuclear morphology, caspase-3 activity, poly-ADP-ribose polymerase cleavage or punctation of mRFP-Bax on mitochondria. Binding of Beclin-1 to Bcl-2 did not modify apoptosis irrespective of Bcl-2 concentration, location or apoptotic stimulus. A similar result was obtained in Atg5-/- cells that are autophagy-deficient, arguing against compensation for the loss of protection by Bcl-2 by autophagy-mediated survival induced by Beclin-1. Hence, although Beclin-1 contains a BH3-only motif typical of pro-apoptotic proteins, it is a negligible modulator of Bcl-2's anti-apoptotic function.

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinised. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.

We reported that administration of anti-tumor necrosis factor alpha (anti-TNF-alpha) antibodies exacerbates the course of a Salmonella infection in both susceptible and resistant mice by preventing the suppression of bacterial growth in the reticuloendothelial system. In the present study, we evaluated the effect of in vivo neutralization of TNF-alpha on the histopathology of primary Salmonella infections. We show that in primary infections, the suppression of bacterial growth in the reticuloendothelial system coincides with granuloma formation in the spleen and liver. Administration of anti-TNF-alpha globulins on day -1 of salmonellosis affected neither the histological picture nor the course of the infection in the early stages of the disease (days 1 to 3), with splenic and hepatic lesions consisting mainly of polymorphonuclear leukocytes (PMNs); conversely, later in infection (days 3 to 7), the treatment inhibited the formation of granulomas. When the anti-TNF-alpha treatment was started well after the suppression of bacterial growth in the reticuloendothelial system and the formation of granulomatous lesions in the spleen and liver, a prompt relapse of the infection and regression of already established granulomas were seen. In anti-TNF-alpha-treated mice, salmonellae were found inside macrophages and PMNs and extracellularly in the necrotic tissue of the spleen, while in the liver the organisms were seen mainly in inflammatory mononuclear cells, resident Kupffer cells, and hepatocytes and occasionally in the extracellular compartment within necrotic lesions. The bacteria appeared most often in clusters, being morphologically intact when in the extracellular space or within hepatocytes, while undergoing various degrees of degeneration when inside phagocytes. The results suggest that TNF-alpha is required for granuloma formation in salmonellosis and that its neutralization does not completely abrogate the bactericidal activity of macrophages and PMNs. Salmonellae were observed to grow within both hepatocytes and phagocytes but were killed only in the latter.

Adenosine 5'-triphosphate (ATP) is known to play a significant role as a neurotransmitter in smooth muscle. There is evidence to show that ATP can cause bladder contractions and may also be involved in the processing of sensory information in the urinary bladder. These effects are likely to be mediated by P2X receptors, namely P2X(1) and P2X(3), respectively. This study set out to investigate their distribution in rat and human urinary bladders. P2X(1) receptor immunoreactivity was found on detrusor muscle fibres and P2X(3) receptor immunoreactivity was found in the urothelium of both species. This is the first demonstration of a non-neuronal localisation for P2X(3) receptors. No clear evidence was found for the presence of P2X(3) receptors on calcitonin gene-related peptide-containing sensory nerves and therefore P2X(3) receptors may not have a direct role in the mediation of sensory responses to ATP in the urinary bladder.

Herpes simplex virus (HSV) nucleocapsids acquire an envelope by budding through the inner nuclear membrane, but it is uncertain whether this envelope is retained during virus maturation and egress or whether mature progeny virions are derived by deenvelopment at the outer nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D (gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with wild-type HSV-1, extracellular virions and virions in the perinuclear space bound approximately equal amounts of gD antibody. In cells infected with HSV-1 encoding an ER-retrieved gD, the inner and outer nuclear membranes were heavily gold labeled, as were perinuclear enveloped virions. Extracellular virions exhibited very little gold decoration (10- to 30-fold less than perinuclear virions). We conclude that the envelope of perinuclear virions must be lost during maturation and egress and that mature progeny virions must acquire an envelope from a post-ER cytoplasmic compartment. We noted also that gD appears to be excluded from the plasma membrane in cells infected with wild-type virus.

The significance of neuronal intranuclear inclusions (NIIs) and extranuclear inclusions (ENNIs) in the brains of patients with polyglutamine repeat diseases and transgenic mice modelling these diseases is hotly debated. We examined inclusions in the brains of mice transgenic for the human Huntington's disease mutation and found that their size, number and location varied markedly with age and neuronal phenotype. In striatum and hippocampus particularly, inclusions appeared at different times in different cell types. Further, the mechanism of formation of inclusions appears to be complex, with several distinct phases. These include a precipitous formation of NIIs followed by NII growth, and the concomitant formation ENNIs. While the timing of appearance of NIIs and ENNIs parallels the cognitive and motor decline of the mice, the precise role of NIIs and ENNIs is unknown. It has been variously suggested that NIIs may be deleterious, benign or beneficial. However, our data allows the possibility that each of these is possible, and suggest also that the role of inclusions changes with time. The precipitous formation of NIIs may play a protective role by removing polyglutamine, while the subsequent growth of NIIs may be deleterious, since it would allow other proteins to be sequestered into inclusions. The formation of ENNIs in neurites and synapses is also more likely to have deleterious than beneficial consequences for a cell. Thus, our study suggests that the relationship between inclusion formation and neurological dysfunction depends not only upon the phenotype of the neurons involved, but also upon the molecular composition and the subcellular localisation of the inclusions.

The effect of loop diuretics at concentrations known to influence cellular water entry coupled to Na-K-Cl co-transport, upon the vacuolation and detubulation following osmotic shock, was investigated in amphibian skeletal muscles. These were exposed to a glycerol-Ringer solution (18 min), an isotonic Ca2+/Mg2+ Ringer solution and cooling. Adding bumetanide (1.0 and 2.0 microM) to these solutions sharply reduced the incidence of detubulation, assessed by abolition or otherwise of action potential after-depolarisations, from 93.9 +/- 4.7% (n = 6) to 5.0 +/- 1.1% (n = 4: mean +/- SEM: 2.0 microM bumetanide). It dramatically reduced the number and fraction of muscle volume occupied by tubular vacuoles, measured using confocal microscopy, from 60.3 +/- 4.3% (n = 10) to 9.0 +/- 1.1% (n = 35). The incidence of large horseradish peroxidase-lined tubular vacuoles, viewed using electronmicroscopy, similarly was reduced with 2 microM bumetanide in the glycerol-Ringer solution. Bumetanide acted through cellular volume adjustments early in the detubulation protocol. Thus, it exerted its maximum effect when added to the glycerol-Ringer, rather than the Ca2+/Mg2+ Ringer solution. Furthermore, whereas fibre diameters measured using scanning electron microscopy returned to normal during glycerol treatment relative to those of control fibres left in isotonic Ringer, addition of 2.0 microM bumetanide in the glycerol Ringer left markedly smaller fibre diameters. Finally equipotent concentrations of the chemically distinct loop diuretics. furosemide and ethacrynic acid similarly influenced detubulation. These findings implicate Na-K-Cl co-transport in the water entry into muscle fibres that would be expected following introduction of extracellular glycerol. This might then enable the subsequent Na-K-ATPase dependent water extrusion that produces the tubular distension (vacuolation) and detachment (detubulation) following glycerol withdrawal, phenomena also observed in muscular dystrophy.

This study examines ion homeostasis in monocyte-macrophages committed to death by apoptosis. X-ray microanalysis has been used to demonstrate that intracellular concentrations of potassium decreased whilst those of sodium increased following 3 h of exposure to 100 microg/ml of oxidized low-density lipoprotein (LDL) in vitro. In contrast, the maximal incidence of cell death, as determined by the inability to exclude trypan blue, was not seen until 24 h of exposure. At 12 h, less than 1 per cent of cells were stained using terminal transferase-mediated DNA nick-end labelling, which is generally accepted as a marker of late stages in the apoptotic pathway. This is the first demonstration of early perturbations of ion homeostasis in monocyte-macrophages exposed to concentrations of oxidized LDL known to cause apoptosis.

It has recently been suggested that the 'vacuolation' of the transverse tubular system that follows the imposition of an osmotic shock is a component process in the eventual 'detubulation' of amphibian skeletal muscle. However, such a hypothesis requires net fluid transfers from the intracellular space into the lumina of the transverse tubules against the prevailing transmembrane osmotic gradients. The present experiments tested the effects of cardiac glycosides on the consequences of established osmotic protocols known reliably to achieve high levels of both detubulation and vacuolation in Rana temporaria sartorius muscle. Tubular isolation (detubulation) was assessed through electrophysiological observations of the abolition or otherwise of the after-depolarisation components of muscle action potentials. Vacuolation was assessed by stereological estimation of the volume fraction of muscle that was occupied by fluorescence-labelled vacuoles observed using confocal microscopy. Introduction of ouabain in the osmotic shock solutions sharply reduced such measures of vacuolation from 48.5 +/- 3.6% (mean +/- SEM; n = 70) to 12.1 +/- 2.7% (n = 190) of the total fibre volume. This was accompanied by sharp reductions in the incidence of detubulation (detubulation index reduced from 96.3 +/- 2.6% to 0.0 +/- 0.0%). The presence of ouabain was critical at the osmotic shock stage in the procedures at which the hypertonic glycerol-containing solutions were replaced by isotonic Ca(2+)-Mg(2+)-Ringer solutions. Finally, the alternative cardiac glycosides, strophanthidine and digoxin, exerted similar effects. These findings support a scheme in which the osmotic shock initiates a metabolically dependent fluid expulsion. This distends the transverse tubules into vacuoles that in turn lead to fibre detubulation.

The cell populations in the dorsal motor nucleus of the vagus (DMNV) of the rat were studied by light microscopy and transmission electron microscopy, including retrograde labeling with horseradish peroxidase and histochemical demonstration of the distribution of the activity of the enzymes acetylcholinesterase (AcChE) and butyrylcholinesterase (BuChE). Two types of neurones were observed: 1) Larger Type A cells, which stain for both AcChE and BuChE and which project into the vagus nerve trunk, and 2) smaller Type B cells, which stain lightly for AcChE but not for BuChE and which do not project into the vagus nerve. Standardised vagal crush at the mid-cervical level causes loss of cholinesterase activity in Type A neurones within a few days but has no effect on Type B neurones. Changes in nuclear morphology of Type A neurones are pronounced at 10 weeks postinjury, indicating that degeneration is irreversible even by this stage. The number of Type A cells projecting to the vagus nerve reduces as a function of time, presumably as these cells die. Only a small number of Type A neurones persist at 2 years postinjury.

Isolated pig hearts, subsequently perfused with pig or human blood, were prepared for the cytochemical demonstration of sites of hydrogen peroxide generation and increased vascular permeability. Oxidant stress was associated with ultrastructural changes commonly seen following myocardial reperfusion. In addition, the precipitation of cerium perhydroxide following perfusion with physiological saline containing cerium chloride suggested the vascular endothelium and leukocytes as sources of oxidants. This was associated with rapid penetration of horseradish peroxidase through the intercellular clefts of the vascular endothelium into the interstitial space, suggesting increased vascular leakiness at these sites. The rapid penetration of horseradish peroxidase was observed at all monitored periods of reperfusion with pig or human blood. This indicates that the increased permeability occurred during the ischaemic period and continued during reperfusion. Morphological damage was greatest in pig hearts reperfused with whole human blood and this was attenuated if the blood was preabsorbed to remove antibodies prior to reperfusion. We conclude that oxidant stress was initiated during ischaemia and continued during reperfusion in this model.

The exposure of amphibian muscle to osmotic shock through the introduction and subsequent withdrawal of extracellular glycerol causes 'vacuolation' in the transverse tubules. Such manoeuvres can also electrically isolate the transverse tubules from the surface ('detubulation'), particular if followed by exposures to high extracellular [Ca2+] and/or gradual cooling. This study explored factors influencing vacuolation in Rana temporaria sartorius muscle. Vacuole formation was detected using phase contrast microscopy and through the trapping or otherwise of lissamine rhodamine dye fluorescence within such vacuoles. The preparations were also examined using electron microscopy, for penetration into the transverse tubules and tubular vacuoles of extracellular horseradish peroxidase introduced following the osmotic procedures. These comparisons distinguished for he first time two types of vacuole, 'open' and 'closed', whose lumina were respectively continuous with or detached from the remaining extracellular space. The vacuoles formed closed to and between the Z-lines, but subsequently elongated along the longitudinal axis of the muscle fibres. This suggested an involvement of tubular membrane material; the latter appeared particularly concentrated around such Z-lines in the electron-micrograph stereopairs of thick longitudinal sections. 'Open' vacuoles formed following osmotic shock produced by extracellular glycerol withdrawal from a glycerol-loaded fibre at a stage when one would expect a net water entry to the intracellular space. This suggests that vacuole formation requires active fluid transport into the tubular lumina in response to fibre swelling. 'Closed' vacuoles only formed when the muscle was subsequently exposed to high extracellular [Ca/+] and/or gradual cooling following the initial osmotic shock. Their densities were similar to those shown by 'open' vacuoles in preparations not so treated, suggesting that both vacuole types resulted from a single process initiated by glycerol withdrawal. However, vacuole 'closure' took place well after formation of 'open' vacuoles, over 25 min after glycerol withdrawal. Its time course closely paralleled the development of detubulation reported recently. It was irreversible, in contrast to the reversibility of 'open' vacuole formation. These findings identify electrophysiological 'detubulation' of striated muscle with 'closure' of initially 'open' vacuoles. The reversible formation of open vacuoles is compatible with some normal membrane responses to some physiological stresses such as fatigue, whereas irreversible formation of closed vacuoles might only be expected in pathological situations as in dystrophic muscle.

When maintaining first trimester placental villi in organ culture under conventional normoxic conditions, we have observed widespread degeneration of the syncytiotrophoblast within 24 h despite excellent viability for the cytotrophoblastic and stromal cell types. Here we identify loss of mitochondrial activity as an early event in this process. In the light of proposals that the early part of gestation occurs in a low oxygen environment and also reported associations between mitochondrial disruption and oxidative stress, we cultured first trimester villi under low oxygen conditions (2.5%). Mitochondrial superoxide dismutase (MnSOD) localization and activity at different gestational ages were also determined. It was found that syncytiotrophoblastic and mitochondrial morphology improved, and mitochondrial activity was retained for 6 h and more if 8- to 10-week-old tissue was placed into a low oxygen environment immediately after removal from the uterus. The effect of oxygen concentration was less marked when using tissue of 14 weeks or more gestational age, which showed good survival and retention of mitochondrial activity under both low and ambient oxygen conditions. This correlated with our finding that placental MnSOD activity increased significantly between 8 and 14 weeks of gestation. Immunohistochemistry demonstrated that at 11 weeks; MnSOD was localized predominantly within the cytotrophoblast cells, whereas by 16 weeks it was found in the syncytiotrophoblast also. These results indicate an acute sensitivity of first trimester placenta syncytiotrophoblast to oxygen-mediated damage.

Steroid-sensitive, vocal courtship behavior is a function of a specific, hypothalamic nucleus, the sexually dimorphic area pars compacta (SDApc) in the male adult gerbil. Gender-related differences in the number of neurons in this nucleus are evident immediately after birth. By using unbiased stereological estimates of cell numbers in Nissl-stained, paraffin-wax sections of brain, we investigated the mechanisms differentiating cell number between the sexes in the SDApc on postnatal days 0, 3, 6, and 15. Cell death, identified by pyknosis, was greatest in the SDApc between days 0-3 in males, whereas in females, maximum values were reached between days 3-6. Similarly, the ratio of pyknotic to normal neurons peaked between days 0-3 in males and 3-6 in females but then declined in both sexes. Pyknotic cells were seldom seen in either sex by day 15. Morphological characteristics of apoptosis including chromatin condensation, cell fragmentation, and ingestion of apoptic bodies by macrophages were all demonstrated by transmission electron microscopy. Macrophages showed specific morphological characteristics of microglia. Cell division (mitosis) was identified in the SDApc during postnatal days 0, 3, and 6 but the numbers of mitotic figures were low, negligible on day 15, and similar between the sexes. These results demonstrate that cell death and proliferation occur simultaneously in the neonatal gerbil brain. The stereological estimates of cell death in the developing SDApc indicated a lower incidence of neuronal death occurring earlier in males than in females.

A characteristic feature of the advanced atherosclerotic lesion is the acellular lipid core, which appears to result at least partly from the death of macrophage foam cells. This study shows that foam cell death at the edge of the lipid core includes both necrosis and apoptosis and that remnants of apoptotic nuclei are present within the lipid core. Apoptotic cells were identified by transmission electron microscopy and by nick end-labelling using terminal deoxynucleotidyl transferase (TUNEL). Some TUNEL-positive cells also expressed proliferating cell nuclear antigen (PCNA). The cause of foam cell death in atherogenesis is unknown, but oxidized low-density lipoprotein (LDL) can cause macrophage apoptosis in vitro and might therefore play a role in the formation and enlargement of the lipid core.

The significance of physical breaches of the trophoblastic layer of the placenta in transmission of HIV from mother to infant was evaluated in 17 HIV-infected pregnant women. Samples of peripheral blood were obtained from the women during pregnancy and at delivery, at which time a small piece of placental tissue was obtained from a random site and immediately placed into fixative. Blood samples were obtained from infants at or shortly after birth and thereafter at approximately 3-month intervals, until the age of 18 months, in order to determine their HIV infection status. HIV RNA and p24 antigen were quantified in maternal plasma and CD4 cells enumerated. Paediatric diagnosis was conducted using polymerase chain reaction, virus isolation, detection of p24 antigen, and measurement of class-specific antibodies. Placental damage was quantified and evaluated using transmission electron microscopy. Maternal viral load was low, with a mean RNA copy number of 8,237 per millilitre of plasma (range 230-37,233 copies/ml). Only two women were p24-antigenaemic, and CD4 numbers ranged from 0.09 to 2.8 x 10(9)/l. There was evidence of breaks in the trophoblastic surface to the depth of the basement membrane in all 17 placentas, and perivillous fibrinoid deposits were also observed to a varying degree in all samples. However, none of the 13 infants available for follow-up had evidence of infection with HIV. Superficial damage to the trophoblastic surface of the placenta, with exposure of the basement membrane and potential exposure of CD4-expressing cells, does not appear to be a significant factor in the transmission of HIV from mother to infant during pregnancy.

This study has demonstrated the toxicity to human monocyte-macrophages of low-density lipoprotein (LDL) which had been artificially oxidized using copper sulphate. The assays of cell damage used were tritiated adenine release, neutral red staining, lactate dehydrogenase leakage, and MTT dye reduction. Toxicity was concentration- and time-dependent. Exposure to native LDL under the same conditions did not result in toxicity. Transmission electron microscopy of cells exposed to oxidized LDL showed characteristic changes of apoptosis, including chromatin condensation and a decrease in cell volume. There was extensive loss of cell surface protrusions and evidence of the phagocytosis of apoptotic cells by neighbouring monocyte-macrophages. Apoptotic features preceded the increased membrane permeability revealed by the release of radioactivity from cells preloaded with tritiated adenine and by lactate dehydrogenase leakage. DNA fragmentation was indicated by nick end-labelling using the terminal transferase enzyme (TUNEL). The number of TUNEL-positive cells was markedly greater in cells exposed to oxidized LDL, compared with those incubated as no-additions controls. Inhibition of de novo protein synthesis with cycloheximide and of Ca2+/Mg(2+)-activated endonuclease activity with aurintricarboxylic acid or zinc ion did not inhibit the toxicity produced by oxidized LDL.

Cell death is a normal feature within the chick chorioallantoic membrane, occurring principally between days 10 and 14 of incubation. Samples of chorioallantoic membrane were obtained on days 6, 8, 10, 12 and 14 of incubation, after the creation of artificial air chambers on day 3. These were examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM) after staining for acid phosphatase activity, and by light microscopy after demonstrating oxygen free radicals with nitro blue tetrazolium. On day 6, small defects in the plasmalemma, approximately 200 nm in diameter, could be seen by SEM. A sequence of events leading to complete destruction of the plasmalemma, with exposure of the nucleus and other cytoplasmic organelles, could be traced, and by day 8 the membrane was a mosaic of healthy cells and others in various stages of degeneration. TEM revealed that acid phosphatase activity was confined to the golgi apparatus and associated vesicles even in advanced stages of degeneration. By comparison, oxygen free radicals were demonstrated in individual cells from day 6 onwards. Application of superoxide dismutase and catalase to the epithelium using a nebuliser spray significantly reduced the amount of cell death seen by scanning microscopy on day 12. It is concluded that oxygen free radicals may mediate cell death in the chorioallantoic membrane.

Tannic acid and dextran have been used to arrest the exocytosis of secretory granules in the atria of the rat heart. By immunogold labeling with silver intensification of ultrathin sections, the arrested exocytosing granules are demonstrated to contain atrial natriuretic peptide (ANP). Extracellular core-like structures found in atria treated with tannic acid are also shown to contain atrial natriuretic peptide. This allows a pathway for the release of atrial natriuretic peptide to be traced from the surface of the myocyte, through the endomysium, into the sub-endothelial space and to the abluminal surface of the capillary endothelium. Uptake of atrial natriuretic peptide into the capillary endothelial cells was also detected. Endothelial transport and release of atrial natriuretic peptide appears to involve the caveolae and smooth vesicles of the non-selective endothelial transport system. No labeling was detected in the endothelial cells of the endocardium or in the mesothelial cells of the epicardium. A disruption of arrested granule cores is seen after dextran treatment. This, and the variation in the labeling of extracellular cores observed, is consistent with the possibility that the cleavage of atrial natriuretic peptide prohormone may be initiated upon the fusion of a granule with the plasma membrane, and continue during the passage of the core material through the extracellular space, with a gradual loss of the active moiety from the disrupted core.

The ultrastructure of cardiac ventricular myocytes isolated from normal and chronically K-depleted rabbits was analysed using morphometric techniques. Myocytes from normal animals showed a quantitatively similar distribution of organelle volumes to those previously reported for isolated myocytes from rat, and for whole myocardium in a variety of species. Myocytes from animals that had been fed a K-deficient diet for 25 days had a significantly increased surface density of junctional sarcoplasmic reticulum with the T-tubules and a significant increase in the surface density of the T-system itself. There were no other differences. The increase in junctional SR and T-system may be related to the defect in cardiac contractility that has been reported for rabbit following K depletion, and may represent an adaptive mechanism acting to protect the heart against Ca overload.

Correlating the surface appearances of certain features with their internal structure is made particularly difficult in the human placenta by the complex three-dimensional branching pattern of the villous tree. This places a possible limitation on the use of the scanning electron microscope in this field, both for basic research purposes and as a tool in pathological diagnosis. To help overcome this problem, a technique for handling individual placental villi has been devised. By attaching single villi to glass pipette tips it has proved possible to scan the villi, embed them in resin and then section them in a known pre-determined orientation. Exact correlations between the surface appearances and the internal structure, as seen with either the light or transmission electron microscope, can then be drawn. This paper describes the technique and, using an example based on syncytial sprouts in early pregnancy, illustrates the precision afforded by the method.

The present investigation was undertaken in rats to determine whether sensory nerves exist in apposition to the bronchial microvessels which may function as rapidly adapting receptors (RAR). The primary and secondary bronchi on both sides were removed and processed for light and electron microscopy. Nerves were frequently found in relation to venules external to the muscle coat of bronchi. They comprised myelinated axons which ended individually as non-myelinated convoluted terminals enclosed within a loose capsule of attenuated cells. Serial sections showed that these terminals were not related to ganglion cells. Cervical vagal section and injection of HRP-WGA into the nodose ganglion provided corroborative evidence of the sensory nature of these terminals. Vagal section caused degenerative changes in the encapsulated nerve terminals in the bronchial walls and horseradish peroxidase labelling was demonstrable in such terminals. Moreover, immunocytochemical studies demonstrated the presence of calcitonin gene regulated peptide and substance P in these structures. It is suggested that they comprise the RAR. Encapsulated nerve terminals were not found in the epithelial layer, in the submucous coat or in the muscularis of bronchi.

Immunocytochemical studies of the adult rat heart show that specific heart granules and atrial natriuretic peptide immunoreactivity are absent from the majority of the myocytes of the specialized nodes, atrioventricular bundle and bundle branches. Immunoreactive granules are present in a small proportion of the transitional sinuatrial and atrioventricular nodal myocytes but, in these regions, they are smaller than their counterparts in the general atrial myocytes. A rarer type of cell profile, identical to general atrial myocytes but lacking immunoreactive granules, is also present at the periphery of the sinuatrial node. A very small proportion of myocytes in the ventricular myocardium, generally in the subendocardial layers subjacent to the terminal ramifications of the bundle branches, contain a few immunoreactive granules.

Ultrastructural studies, including a preliminary morphometric analysis, of the right ventricular myocardium in the West African bat Eidolon helvum show that the myocytes contain a wider T-tubule system and a higher proportion of mitochondria and lipid droplets than in typical terrestrial mammals such as the rat; these features in the bat are even mor pronounced than in hibernating species such as the golden hamster. In addition to having coupling arrangements with T-tubules and surface sarcolemma, cisterns of junctional sarcoplasmic reticulum are closely apposed to mitochondria and lipid droplets.

The atrioventricular (AV) node of the golden hamster is situated unusually high in the interatrial septum when compared to other species such as the rat. 2 main cell types, characterized by electron-lucent or electron-dense cytoplasm respectively, are found in the node; although both types contain numerous myofilaments these are irregularly arranged and sarcomeric banding is poor. A third variety comparising transitional cells, with features intermediate between the main nodal cells and general atrial myocardial cells, are found at the periphery of the node. Similar electron-lucent and electron-dense cells are also found in the bundle and the mean diameter of bundle cells increases as one passes from the node to the bundle bifurcation. In the node, specific heart granules (SHG) identified by ANP-28 immunoreactivity are found only in transitional cells and even here they are very sparse, unlike general atrial myocytes in which they are plentiful. Numerous nerve varicosities are present throughout the node and bundle and 5-hydroxydopamine (5-OHDA) labelling demonstrates that most of them have features of either noradrenergic or cholinergic terminals; a few non-cholinergic, non-adrenergic varicosities are also present.

The differentiation of the myocardial rudiment was examined in mouse embryos, isolated between the afternoons of the eighth and ninth days of gestation, by means of transmission electron microscopy of ultrathin sections and of freeze-fracture replicas; some of the material used for sectioning was labelled with ruthenium red. Formation of the presumptive pericardial cavity commences during the late presomite stage (afternoon of the eighth day) and the myocardial rudiment originates in situ as a thickening of the splanchnic pericardial lining. Initially, the myocardium comprises an epithelium or plate directly exposed to the pericardial lumen and overlying a separate layer of endocardial elements. As the heart tube bulges into the pericardial coelom, it becomes surrounded by a sleeve of myocardium which thickens and stratifies during the ninth day and subsequently (on the tenth day) acquires an epicardial covering of flattened cells. The myocardium commences pulsations at or about the 3-4 somite stage (morning of the ninth day) by which time the myoblasts already contain striated myofibrillae and specialised cell junctions. From its earliest appearance, the myocardial plate contains tight junctions and desmosomes between the lateral borders of the apical parts of the myoblasts. Gap junctions soon appear in the same regions and they increase in number and extent, as the myoblasts elongate and divide, thus establishing contact in various planes; they are supplemented by the formation of fasciae adherentes and of more desmosomes. On the other hand, tight junctions decline in extent and are eventually confined to the epicardium. Other features such as caveolae and T-tubules are not established by the end of the ninth day and coupling arrangements of the sarcoplasmic reticulum are only in the rudimentary stages of formation.

Atrial and ventricular myocardium from young golden hamsters, 3-6 months of age, was examined under the transmission electron microscope, representative samples from each animal having been stained histochemically for acid phosphatase (AP) by a lead precipitation technique. Our observations indicated that AP positive primary lysosomes are produced at the sites of Golgi-endoplasmic reticulum-lysosome (GERL) complexes and the main pathway for lipofuscin production is by fusion of primary lysosomes with mitochondria in both atrial and ventricular myocytes. The mitochondria are then progressively degraded, their matrix becomes denser and there is an accompanying increase of AP activity. After reaching a peak level, AP activity declines until an AP negative terminal pigment body with a smooth outline is produced. Another pathway, but much less frequent at this age leads to the formation of larger bodies, irregular in outline and containing small globules. Their genesis entails the participation of autophagic vacuoles which engulf lipid droplets, mitochondria or amorphous debris. Like the main variety of lipofuscin body, they contain AP while digestion progresses but eventually become AP negative. A few lipofuscin granules are eliminated by being pinched off within fine cell protrusions and are subsequently engulfed by scavenger cells.

There was no statistically significant difference in basal concentrations of immunoreactive atrial natriuretic peptide (ANP), as assessed by radioimmunoassay, between right and left atrial muscle of control rats; similarly, stereological analysis showed no statistically significant difference in the fractional volume of myocytes occupied by specific heart granules, or in numerical density of granules, between right and left atria. Nevertheless, correlated radioimmunoassay and ultrastructural investigations showed that the major source of elevated plasma levels of ANP after expansion of blood volume was the right atrium. Substantial expansion of blood volume caused an increase in the proportion of peripherally located granules in myocytes of both atria, but reduction in the number of granules and in the concentration and total content of ANP occurred in the right atrium only. Bilateral cervical vagotomy also caused a statistically significant elevation of plasma ANP concentration, accompanied by a statistically significant reciprocal reduction in right atrial ANP content; no statistically significant change occurred in left atrial ANP. When blood volume was expanded after bilateral vagotomy, there was a further statistically significant increase in plasma ANP concentration; this was accompanied by further reduction in right atrial ANP and, moreover, the combined manoeuvre also elicited a statistically significant reduction of ANP in the left atrium. Ultrastructural studies confirmed that, under these conditions, myocytes in both atria showed a marked depletion of specific heart granules.

The ontogenesis of specific heart granules and of the related natriuretic peptide activity in heart muscle was studied in fetal and neonatal rats and golden hamsters by ultrastructural analysis including immunogold labelling for ANP-28 and by radioimmunoassay. In both species, immunoreactive granules first appear in the myocardial sleeve of the embryonic heart tube during the looping stages which precede chamber formation and the peptide becomes detectable by radioimmunoassay two or three days later by which time the chambers are identifiable. Granule density and ANP concentration in the rat are higher than in the hamster at all stages of development. Almost all atrial myocytes express ANP in fetal hearts whereas, in the ventricular wall, cells containing immunoreactive granules are scattered. The density of granules in atrial myocytes increases during further stages of fetal and neonatal development, while it decreases markedly even in those ventricular myocytes which are immunoreactive. Changes in the ultrastructural appearance of ventricular SHG suggest that the mode of production of ANP changes in ventricular myocytes after birth but does not change in atrial cells. There is no correlation between the distribution of immunoreactive ventricular myocytes and that of the conducting system. In both species, the concentration of ANP in the atrial well is higher than ventricular levels from the outset and the disparity becomes exaggerated with development till, in six months old adult animals, the atrial to ventricular concentration ratio is about 3 x 10(3):1 in the rat and 1.5 x 10(3): 1 in the hamster. In the hamster, a distinct gradient of ANP concentration between the right and left atria is already established in the early fetal period and it becomes enhanced in the neonatal period. In the rat, however, a slight difference becomes discernible only after birth.